Constructs for enhancing immune responses

ABSTRACT

Chimeric protein constructs including a herpesvirus glycoprotein D (gD) and a heterologous polypeptide that interact with herpes virus entry mediator (HVEM) and enhance and enhance an immune response against the heterologous polypeptide and methods for their use are provided.

This application is a continuation of Ser. No. 12/438,889 filed on Feb.25, 2009. Ser. No. 12/438,889 is a National Stage application ofPCT/US2007/018939 filed on Aug. 28, 2007, which claims the benefit ofand incorporates by reference Ser. No. 60/840,526 filed Aug. 28, 2006.

This invention was made with government support under 5 P01 AI052271awarded by National Institutes of Health. The government has certainrights in the invention.

This application incorporates by reference the contents of a 63.9 kbtext file created on Aug. 1, 2011 and named“00048600037sequencelisting.txt,” which is the sequence listing for thisapplication.

FIELD OF THE INVENTION

Embodiments of the present invention relate in general to chimeric(fusion) protein constructs including a herpesvirus glycoprotein D (gD)and a heterologous polypeptide (e.g., antigen) that enhance the immuneresponse against the heterologous polypeptide (e.g., antigen) in asubject.

BACKGROUND OF THE INVENTION

gD is the receptor-binding glycoprotein of herpesviruses (Fusco et al.(2005) Proc. Natl. Acad. Sci. U.S.A. 102:9323). The gD ectodomain isorganized in two structurally and functionally differentiated regions.The amino-terminus includes the signal sequence and receptor-bindingsites, and the carboxy-terminus includes the pro-fusion domain and thetransmembrane domain. gD interacts with two alternative receptorsbelonging to unrelated protein families, the herpesvirus entry mediator(HVEM) and the nectins (Geraghty et al. (1998) Science 280:1618;Montgomery et al. (1996) Cell 87:427: Cocchi et al. (1998) J. Virol.72:9992; Warner et al. (1998) Virology 246:179; Lopez et al. (2000) J.Virol. 74:1267). HVEM is expressed on dendritic cells and the B and Tlymphocyte attenuator (BTLA) is expressed on activated T and Blymphocytes. The interaction between HVEM and BTLA results in thedown-regulation of immune responses.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a schematic representation of the chimeric gene gDE7E6E5.The HPV-16 E5, E6 and E7 genes without respective start and stop codonswere linked in tandem and incorporated into the HSV-1 gD gene ApaI site,which corresponds to amino acid 244 in the gD mature form.

FIG. 2 depicts a schematic representation of chimeric gene gDgag. Thecodon-optimized truncated form of gag from HIV-1 clade B was fused intothe HSV-1 gD gene NarI site, which corresponds to amino acid 289 in thegD mature form.

FIGS. 3A-3B depict the gag-specific CD8⁺ IFN-γ response in miceimmunized with vaccine constructs carrying the gDgag chimeric gene. FIG.3A depicts a FACS analysis of the gag-specific CD8/IFN-γ response inperipheral blood mononuclear cells (PBMC) from mice immunized with DNAvaccine expressing either HIV-1 gag or HIV-1 gag fused to HSV-1 gD(gDgag). Numbers on the right corner represent percentage CD8⁺/IFN-γ⁺cells over total of CD8⁺ cells. FIG. 3B graphically depicts PBMC frommice immunized with AdC68 vectors carrying either genes encoding gag orgDgag, inoculated with different amounts of virus particles per mouse.

FIG. 4 graphically depicts the effect of pre-existing immunity to theAdHu5 adenovirus vector on the transgene product-specific CD8⁺ T cellresponse to the AdC68 vector. AdC68 vectors carrying gag, gDgag orgDE7E65 were inoculated into naïve mice and mice previously immunizedwith an AdHu5 expressing an unrelated antigen (rabies glycoprotein,AdHu5rab.gp). The percentage of reduction of CD8⁺ T cell response wasdefined as a percentage of CD8⁺/IFN-γ⁺ frequency in mice previouslyimmunized with AdHu5 over the frequency found in mice that did notreceive AdHu5 vector.

FIG. 5 depicts the E7-specific CD8⁺ IFN-γ response in mice immunizedwith DNA vaccines expressing non-mutated or mutated gDE7E6E5. Themutation was designed to disrupt the HVEM binding site of gD. Mice wereimmunized with non-mutated gDE7E6E5 (pgDE7E6E5) or mutated gDE7E6E5(pNBEFgDE7E6E5) and 14 days later peripheral blood mononuclear cellswere investigated by intracellular cytokine staining for E7-specificCD8⁺ IFN-γ responses. Numbers on the right corners represent percentageCD8⁺/IFN-γ⁺ cells over total of CD8⁺ cells.

FIG. 6 depicts the E7-specific CD8⁺ IFN-γ response in mice immunizedwith DNA vaccines expressing non-mutated (gDE7) or mutated gD (SgDE7).This mutation (SgD) was designed to increase binding between gD andHVEM. Mice were immunized with pgDE7 or pSgDE7 and 14 days laterperipheral blood mononuclear cells were investigated by intracellularcytokine staining for E7-specific CD8⁺ IFN-γ response. Numbers in thecorners represent percentage CD8⁺/IFN-γ⁺ cells over total of CD8⁺ cells.

FIG. 7 graphically depicts an in vitro HVEM binding assay. CHO-CAR cells(Chinese hamster ovary-coxsackie-adenovirus receptor cells) wereinfected with either AdC68gD or AdC68gDgag and, after 48 hours, totalprotein was extracted. The amount of gD in each sample was quantified bycapture ELISA and the protein extracts were diluted in extraction bufferto normalized levels of gD. Equalized extracts were diluted and added to96-well plates coated with purified HVEM. The amount of gD bound to HVEMwas detected by using anti-gD polyclonal antisera and anti-Rabbit IgGhorseradish peroxidase. Data shown is one representative experiment fromtwo performed.

FIGS. 8A-8B depict confocal microscopy for localization of gDgag andHVEM on AdC68gDgag infected cells. B78H1/3E5 cells, which expressed HVEMfused to enhanced green fluorescence protein (HVEM-EGFP), were infectedwith AdC68gDgag. After 48 hours, cells were either directly stained(FIG. 8A) or permeabilized and stained (FIG. 8B) with an anti-gDmonoclonal antibody (DL-6) and anti-mouse IgG conjugated with Texas Red.Cells were examined with a Leica TCS SP2 Confocal Microscope at 400×final magnification.

FIG. 9 graphically depicts FACS analysis of gDgag expression on thesurface of AdC68gDgag infected cells. B78H1/3E5 cells (darker line),which express HVEM-EGFP on the surface, and B78H1 cells (lighter line),which do not express HVEM, were infected with AdC68gDgag. Cells werecultivated for 48 hours, and then labeled with an anti-gD monoclonalantibody (DL-6) and anti-mouse IgG conjugated to phycoerythrin (PE).Cell suspensions were analyzed using an EPICS XL (Beckman-Coulter, Inc.,Miami, Fla.) to determine presence of gDgag.

FIGS. 10A-10B depict that gDgag expressed by infected cells bound toHVEM expressed on non-infected cells. Confocal microscopy (FIG. 10A) andFACS analysis (FIG. 10B) were performed to localize gDgag onnon-infected cells. CHO-CAR cells were infected with AdC68gDgag. After48 hours, cells were harvested and washed extensively with cold PBS.AdC68gDgag-infected CHO-CAR cells were cultured with B78H1/3E5 cells,which expresses HVEM-EGFP on the surface, at 4:1 ratio. After 48 hours,cells were stained using the anti-gD monoclonal antibody DL-6 andanti-mouse IgG conjugated to Texas Red (microscopy) or PE (FACS). (FIG.10A) Microscopy was performed with a Leica TCS SP2 Confocal Microscopeat 400× final magnification. (FIG. 10B) Cell suspensions were analyzedusing an EPICS XL (Beckman-Coulter, Inc., Miami, Fla.). B78H1/3E5 cellswere cultured with either AdC68gDgag-infected CHO-CAR cells (darkerline) or non-infected CHO-CAR cells (lighter line). Data on graph showcells which are positive for GFP.

FIG. 11 depicts the gag-specific CD8⁺ T cell response in mice immunizedwith AdC68 vectors. PBMC and splenocytes from mice immunized with 1×10⁹vp of AdC68 vectors carrying either gag, gDgag or gD were tested.Percentage represents CD8⁺/IFN-γ⁺ cells over total of CD8⁺ cells.CD8⁺/IFN-γ⁺ frequencies in all groups stimulated with an unrelatedcontrol peptide were below 0.20%. Data shown are representative of twoperformed experiments.

FIG. 12 graphically depicts the gag-specific IFN-γ response of CD8⁺ Tcells from mice immunized with AdC68 vectors. PBMC are from miceimmunized with different amounts of AdC68 vectors carrying either gag,gDgag or gD. Percentage represents CD8⁺/IFN-γ⁺ cells over total of CD8⁺cells. CD8⁺/IFN-γ⁺ frequencies in all groups stimulated with anunrelated control peptide were 0.20%. Data shown are representative oftwo performed experiments.

FIG. 13 depicts the phenotypic profile of CD8⁺ cells activated by AdC68vaccination. PBMC from naïve mice and mice immunized with AdC68 carryingeither gag or gDgag were stained with gag-tetramer-APC andanti-CD8-PerCP, in combination with anti-CD25-PE, anti-CD-122-PE,anti-CD127-PE, anti-CD27-PE, anti-CD62L-FITC, anti-CD69-PE,anti-CD103-PE, anti-CD43-PE, anti-CD44-FITC, anti-CD54-PE, anti-Bcl2-PE,anti-BTLA-PE, anti-CTLA4-PE and anti-PD1-PE. Graphs show data fromCD8⁺/gag-tet⁺ cells for AdC68gag (gray line) and AdC68gDgag (blackline), and total CD8⁺ for naïve (black dotted line). Data were analyzedon Flowjo software (Tree Star Inc.).

FIG. 14. Intracellular IFN-γ staining of gag-specific and AAV-specificCD8⁺ T cells from mice immunized with AAV vectors expressing gD, gag orgDgag. Detection of gag-specific and AAV-specific CD8⁺ T cells wascarried out after stimulation of peripheral blood mononuclear cells(PBMCs) with either MHC class I restricted gag peptide or AAV-capsidpeptide and cell surface staining for CD8 and intracellular staining forIFN-γ. Unspecific peptide was used as control. The numbers in the rightupper corners show the frequencies of peptide-specific CD8⁺ T cells, aspercentages of IFN-γ-producing CD8⁺ T cells over all detected CD8⁺ Tcells.

FIG. 15. Schematic representation of chimeric gene gD-NP. NucleoproteinP (NP) from Influenza virus A/PR8 without its start and stop codons wasincorporated into the HSV-1 gD ApaI site, which corresponds to aminoacid 244 in the mature form of gD.

FIG. 16. NP-tetramer staining of CD8⁻ T cells isolated from blood ofmice immunized with either pgD-NP or pNP DNA vaccine. Mice wereimmunized with 100 μg of each DNA vaccine vector. Fourteen days afterimmunization peripheral blood mononuclear cells (PBMCs) were isolatedand cell surface stained with the NP-tetramer and a labeled antibody toCD8. Naïve mice were used as negative control. Data representpercentages of NP-tetramer⁺ CD8⁺ T cells over all detected CD8⁺ T cells.

FIG. 17. Schematic representation of chimeric gene gDTRAPTB.Thrombospondin-related anonymous protein (TRAP) from parasite Plasmodiumfalciparum and Mycobacterium tuberculosis epitope string (TB) withouttheir start and stop codons are incorporated into the HSV-1 gD NarIsite, which corresponds to amino acid 288 in the gD mature form.

FIGS. 18A-D. Molecular modeling of the gD-gag chimeric protein. Ribbonrepresentations of gD-gag in the unligated (FIGS. 18A, 18B) and HVEMligated (FIGS. 18C, 18D) conformations. FIG. 18A, the gag insertion isconnected to the gD ectodomain core by a long flexible linker. FIG. 18B,a superposition of the native gD X-ray structure (2C36) (darker ribbon)and that of the gD-gag chimera model (lighter ribbon) shows that the gaginsert repositions the C-terminus of native gD away from the HVEMbinding pocket. The dashed line indicates an 11 residue gD loop segmentthat like the first 22 N-terminal residues (not shown) is unresolved inthe X-ray structure and presumed to be highly flexible (Krummenacher etal., EMBO J. 24, 4144-53, 2005). FIG. 18C, gD-gag chimera model in theHVEM ligated conformation with HVEM positioned as observed in thegD-HVEM complex X-ray structure (1JMA). The gD N-terminus changesconformation upon formation of the HVEM complex. FIG. 18D, asuperposition of the native gD X-ray structure (2C36) (darker ribbon)with the gD-gag chimera model (lighter ribbon) in the HVEM-boundconformation shows that the gag insert does not disrupt the gD coredomain.

FIG. 19. Confocal microscopy was carried out with B78-H1/3E5 cells,which express HVEM fused to Enhanced Green Fluorescence Protein(HVEM-EGFP). B78-H1/3E5 cells were infected with AdC68gag, AdC68gD orAdC68gD-gag, then stained anti-gD DL-6 MAb and anti-mouse IgG conjugatedwith Texas Red. AdC68gD-gag-infected cells were permeabilized thenstained as above. Cells were examined with a Leica TCS SP2 ConfocalMicroscope at 400× final magnification.

FIG. 20. Comparison of gD expression on the surface of B78-H1 (blackline) and B78-H1/3E5 (gray) cells infected with AdC68 vectors carryinggD-gag, gD or gag.

FIG. 21. Presence of gD on the surface of non-infected HVEM⁺ cellsco-cultivated with HVEM⁺ cells infected with either AdC68gD-gag, AdC68gDor AdC68gag. Non-infected cells were used as negative control.

FIG. 22. AdC68gD induces enhanced expansion of CD8⁺ T cells in vitro.Irradiated lymph nodes cells from naïve mice and mice immunized witheither AdC68E7E6E5 or AdC68gD were incubated with CFSE-labeled CD8⁺ OT-1(Vα2⁺) cells for 72 hrs. Total live cells (left) were analyzed forexpression of CD8⁺ Vα2⁺. CSFE expression by these double positivepopulations (highlighted by the squares on the right graphs) is shown onthe left graphs. The bars and numbers show from right to left thepercentages of the population that underwent no replication, 1, 2, 3 or≦4 cycles of replication. Graphs show data from one representativeexperiment of two performed.

FIGS. 23A-C. CD8⁺ T cell responses to vectors expressing antigens fusedto gD. FIG. 23A, intracellular cytokine staining of E7- and gag-specificCD8⁺ T cells were carried out on PBMCs from mice i.m. immunized with DNAvaccines (upper graphs) or AdC68 vectors (lower graphs) expressingeither gD, E7E6E5, gD-E7E6E5, gag or gD-gag, after stimulation with E7or gag peptide and cell surface staining for CD8 (FITC) andintracellular staining for IFN-γ (PE). PBMCs were isolated from animals14 days after DNA vaccination or 10 days after application of AdC68vector. The numbers in the right upper corners show frequencies ofIFN-γ-producing CD8⁺ T cells as a percentage of all CD8⁺ T cells.Frequencies of IFN-γ⁺/CD8⁺ T cells stimulated with an unrelated controlpeptide were below 0.2% in all groups. FIG. 23B, Gag-specific CD8⁺ Tcell frequencies were determined 10 days after immunization of mice withdecreasing doses of either AdC68gag (open bars) or AdC68gD-gag (blackbars) vectors. FIG. 23C, The kinetics of E7-specific CD8⁺ T cellresponses induced by the AdC68gD-E7E6E5 vector were analyzed from BPMCsof mice immunized with either AdC68E7E6E5 (squares), AdC68gD (diamonds)or AdC68gD-E7E6E5 (triangles) vectors at different days after a singledose of 10¹⁰ vps of the vaccines.

FIGS. 24A-B. The enhancement of CD8⁺ T cell responses requires bindingof gD to HVEM. FIG. 24A, E7-specific IFN-γ⁺CD8⁺ responses were evaluatedwith splenocytes from mice immunized with one dose of DNA vaccinesexpressing the E7E6E5 polypeptide either within wild-type gD(pgD-E7E6E5), a mutated gD that shows loss of binding to HVEM(NBEFgD-E7E6E5) or that shows enhanced binding to HVEM (SgD-E7E6E5).FIG. 24B, splenocytes from mice immunized with one dose of DNA vaccinescarrying E7 fused to either wild type gD (gD-E7) or mutated gD with highaffinity to HVEM (SgD-E7) were evaluated for E7-specific IFN-γ⁺CD8⁻response. PBMCs were isolated 14 days after DNA vaccine immunizations.

FIG. 25. Phenotypes of gag-specific CD8⁺ T cells were analyzed on PBMCsfrom mice immunized with either AdC68gD-gag or AdC68gag. PBMCs wereisolated 10 days after immunization. Naïve mice were used as controls(black dotted line). The graphs shown reflect expression levels of totalCD8⁺ T cells from naïve mice (black dotted line) and gag-tet⁺ CD8⁺ Tcells from mice immunized with either AdC68gag (grey line) orAdC6SgD-gag (black line).

FIGS. 26A-C. CD8⁺ T cells induced by gD-antigen chimeric protein arefunctional in vivo. Protection against TC-1 tumor challenge wasevaluated in mice vaccinated with DNA (FIG. 26A) or AdC68vectors (FIG.26B) expressing either gD (circles), E7E6E5 (diamonds) or gD-E7E6E5(squares). FIG. 26C, Protection to TC-1 tumor challenge in micevaccinated with DNA vaccine expressing either NBEFgD-E7E6E5 (diamonds),SgD-E7E6E5 (squares), gD-E7 (circles) or SgD-E7 (triangles) chimericgenes. Mice were challenged 14 and 10 days after vaccination with DNAand AdC68 vectors, respectively. Tumor development was followed for upto 60 days after challenge.

FIG. 27A-B. Quantification of specific mRNA copies and proteinexpression by cells infected in vitro with AdC68 vectors. FIG. 27A, RNAisolated from non-infected cells and from cells infected with AdC68gD(white bars), AdC68E7E6E5 (gray bars) or AdC68gD-E7E6E5 (black bars)were reverse transcribed and quantified by Real-Time PCR. Afterquantification of GAPDH mRNA copies, all samples were normalized to 109GAPDH mRNA copies. Specific mRNA copies were quantified using gD, E7,E6, and E5 specific primers. Neither E7, E6 nor E5 mRNA were detected incells infected with AdC68gD, and no gD specific mRNA was detected incells infected with AdC68E7E6E5. mRNA levels were assessed in threeindependent experiments and each sample was investigated in triplicates,p values from two-tail student's t test are shown on top of the bars.FIG. 27B, confocal microscopy was carried out with CHO/CAR cellsinfected with AdC68 expressing either gD, E7E6E5 or gD-E7E6E5, thenpermeabilized and stained with anti-gD DL-6 MAb and anti-mouse IgGconjugated with FITC. Immunofluorescence is shown on the top panel whiledifferential interference contrast (DIC) microscopy is shown on thebottom panel. Cells were examined with a Leica TCS SP2 ConfocalMicroscope at 400× magnification.

FIG. 28A-C. Intracellular IFN-γ staining of E7-specific CD8⁺ T cellsfrom mice immunized with AdC68 and DNA vectors. FIG. 28A, frequencies ofE7-specific CD8⁺ T cells in PBMC (top) or spleens (bottom) from naïvemice or mice immunized with AdC68 vector expressing either gD, E7E6E5 orgD-E7E6E5 were determined 10 days after immunization. FIG. 28B,frequencies of E7-specific CD8⁺ T cells in PBMC (top) or spleens(bottom) from naïve mice or mice immunized with DNA vaccines expressingeither gD, E7E6E5 or gD-E7E6E5 were determined 14 days afterimmunization. FIG. 28C, prime and boost regimens with pgD-E7E6E5 andAdC68gD-E7E6E5 vectors. Mice immunized with one dose of pgD-E7E6E5vector were boosted after 90 days with AdC68gD-E7E6E5 (open bars), whilemice immunized with AdC68gD-E7E6E5 were boosted after 90 days withpgD-E7E6E5 (black bars). Detection of E7-specific CD8⁺ T response wascarried out after stimulation with a MHC class 1 restricted E7 peptideand cell surface staining for CD8 (FITC) and intracellular staining forIFN-γ (PE). The numbers in the right upper corners show the frequenciesof E7-specific CD8⁺ T cells, as percentages of IFN-γ-producing CD8⁺ Tcells over all detected CD8⁺ T cells. IFN-γ-producing CD8⁺ cellfrequencies in all groups stimulated with an unrelated peptide or in theabsence of stimulus were below 0.2%. The data shown in a and b are fromone representative experiment of four performed.

FIG. 29. Dose-response of the CD8⁺ T cell response to AdC68gD-E7E6E5.Frequencies of IFN-γ-producing E7-specific CD8⁺ T cells in spleens andPBMCs induced by 5×10¹⁰ to 1×10⁸ vp/animal of AdC68gD-E7E6E5 vector weredetermined as described on legend to FIG. 28.

FIG. 30A-B. Anti-tumor effects of AdC68 vectors against TC-1 cellchallenge. FIG. 30A, for post-challenge vaccination, groups of 10 micewere s.c. inoculated with TC-1, then 5 days later immunized with eitherAdC68gD (triangles), AdC68E7E6E5 (circles) or AdC68gD-E7E6E5 (squares).FIG. 30B, for pre-challenge vaccination, groups of 10 mice vaccinatedone year earlier with AdC68 vectors carrying either gD (triangles),E7E6E5 (circles) or gD-E7E6E5 (squares) were s.c. challenged with TC-1.For all TC-1 challenge experiments animals were monitored 3 times perweek for evidence of tumor growth over a period of 60 days.

FIG. 31A-B. CD8⁺ T cell response after challenge with TC-1 in micevaccinated one year earlier. One year after vaccination with AdC68vectors expressing either gD, E7E6E5 or gD-E7E6E5, mice were challengedwith TC-1 cells and 10 days later E7-specific frequencies of E7-specificCD8⁺ T cells were determined. FIG. 31A, ICS of E7-specific CD8⁺ T cellsin spleen from non-challenged mice (top) or mice challenged with TC-1cells (top). Frequencies of IFN-γ-producing E7-specific CD8⁺ T cells inspleen were determined as in FIG. 2 legend. FIG. 31B, E7-tetramerstaining of CD8⁺ T cells isolated from spleen, blood and liver of micechallenged (white bars) or not (black bars) with TC-1 cells. Datarepresent percentages of E7-tetramer⁺ CD8⁺ T cells over all detectedCD8⁺ T cells. E7-tetramer⁺ CD8⁺ cells were not detected in miceimmunized with either AdC68gD or AdC68E7E6E5.

FIG. 32A-C. CD8⁺ T cell response and phenotypic profile in miceimmunized with AdC68 vectors and subsequently challenged with TC-1cells. Lymphocytes were isolated 3 days after TC-1 challenge fromanimals immunized with AdC68gD-E7E6E5 and 7 days after challenge fromanimals immunized with either AdC68gD or AdC68E7E6E5. E7-specific CD8⁺ Tresponse were determined in spleen, PBMC and TIL by ICS (FIG. 32A) andE7-tetramer staining (FIG. 32B). ICS data was determined as in FIG. 2legend, while E7-tetramer staining data represent percentages ofE7-tetramer⁺ CD8⁺ T cells over all detected CD8⁺ T cells. E7-specificIFN-γ CD8⁺ cells and E7-tetramer⁺ CD8⁺ cells were not detected in miceimmunized with either AdC68gD or AdC68E7E6E5. FIG. 32C, phenotypeanalysis of splenocytes, PBMCs and TILs were determined with cellsisolated from mice immunized with AdC68gD-E7E6E5 (black line) or AdC68gD(dotted black line) then challenged with TC-1. Cells were stained withE7-tetramer-APC and anti-CD8-PerCP, in combination with antibodies toCD44, CD62L, CD27, Bcl2, BTLA, CTLA-4 and PD-1. CD8⁺ T cells isolatedfrom either naïve mice or mice immunized with AdC68E7E6E5 showed similarphenotype profiles as CD8⁺ T cells isolated from mice immunized withAdC68gD.

FIG. 33A-C. Comparison of CD8⁺ T cell responses and phenotype profilesinduced by AdC68 vectors in wild-type and HPV-16 E6/E7-tg mice. One-yearold E6/E7-tg mice were vaccinated with AdC68 vectors expressing eithergD, E7E6E5 or gD-E7E6E5, and 1-year old C57Bl/6 mice were vaccinatedwith AdC68gD-E7E6E5 vector. Ten days later frequencies and phenotypes ofE7-specific CD8⁺ T cells were determined. FIG. 33A, E7-specific CD8⁺ Tcells isolated from spleens of E6/E7-tg mice immunized with AdC68gD,AdC68E7E6E5 or AdC68gD-E7E6E5, or from spleen of wild-type miceimmunized with AdC68gD-E7E6E5 were tested by ICS. Frequencies ofIFN-γ-producing E7-specific CD8⁺ T cells in spleen were determined asdescribed in legend to FIG. 2. FIG. 33B, E7-tetramer staining of CD8⁺ Tcells was performed with splenocytes and PBMC from wild-type andE6/E7-tg mice, and with lymphocytes from thyroids of E6/E7-tg mice. Datashow percentages of E7-tetramer⁺ CD8⁺ T cells over all detected CD8⁺ Tcells. ND, not determined. FIG. 33C, phenotypic profile of E7-specificCD8⁺ T cells were determined using cells isolated from spleen and bloodof AdC68gD-E7E6E5 vaccinated E6/E7 tg (filled gray) and wild-type (blackline) mice, and from cells isolated from thyroid of E6/E7 tg micevaccinated with AdC68gD-E7E6E5. CD8⁺ T cells isolated from E6/E7 tgnaïve mice were used as controls (black dotted line).

DETAILED DESCRIPTION

The invention provides chimeric, or fusion, proteins in which one ormore antigens is inserted into the C terminal region of a mature HSV gDprotein. Such fusion proteins enhance the immune response of a hostagainst the antigen(s) to a much greater degree than is observed withoutthe gD. The gD chimeric proteins of the present invention areparticularly suitable for use as genetic vaccines (e.g., DNA vaccines orviral vector vaccines) to therapeutically or prophylactically treat asubject. Thus, the invention also provides nucleic acid molecules whichencode fusion proteins of the invention.

Glycoprotein D

Glycoprotein D (gD) is an envelope glycoprotein found on Herpes simplexviruses such as HSV-1 or HSV-2 and is expressed in cells infected by theviruses. An HSV gD has a 25-amino acid amino-terminal signal sequenceand a carboxy-terminal transmembrane domain. The signal sequence istypically cleaved in the mature form of the protein. The amino acidsequence of HSV-1 gD is shown in SEQ ID NO:27 (amino acids 1-25 are thesignal sequence; amino acids 26-394 are mature HSV-1 gD); a codingsequence for SEQ ID NO:27 is shown in SEQ ID NO:26. The amino acidsequence of HSV-2 gD is shown in SEQ ID NO:29 (amino acids 1-25 are thesignal sequence; amino acids 26-393 are mature HSV-2 gD); a codingsequence for SEQ ID NO:25 is shown in SEQ ID NO:28.

An HSV gD or mutant thereof which is useful in the present invention hasthe ability to interact with HVEM and, in addition, may have one or moreof the following properties: 1) ability to stimulate a CD8⁺ T cellresponse to the fusion partner; 2) ability to disrupt an HVEM-BTLApathway activity; 3) ability to interact with nectin-1; 4) ability tomediate cell entry by an HSV-1 and/or HSV-2 virus; and 5) ability tomediate cell-to-cell spread of HSV-1 and/or HSV-2. Thus, as used herein,a “gD” or an “immunostimulatory portion of a gD” refers to a polypeptidehaving an amino acid sequence of a wild-type gD or a mutant thereofwhich retains one or more gD activities.

gD chimeric (fusion) proteins of the invention comprise at least two,preferably three polypeptide segments. The first polypeptide segmentcomprises at least amino acids 1-240 of a mature Herpes simplex virus(HSV) glycoprotein D; in preferred embodiments the first polypeptidesegment does not comprise a full length mature glycoprotein D; in thiscase a third polypeptide segment is included. The second polypeptidesegment, the N terminus of which is linked to the C terminus of thefirst polypeptide segment, comprises at least one antigen which is notan HSV glycoprotein D antigen. The third polypeptide comprises a Cterminal portion of the HSV glycoprotein D; the N terminus of the thirdpolypeptide segment is linked to the C terminus of the secondpolypeptide segment. Thus, in certain embodiments an antigen is fused tothe carboxy-terminal region of gD. In other aspects, an antigen isinserted within the carboxy-terminal amino acid sequence of gD such thatthe amino-terminal end of the gD chimeric protein is an amino-terminalgD amino acid sequence, fused to an internal antigenic sequence, fusedto a carboxy-terminal amino acid sequence of gD.

In certain embodiments, the first polypeptide segment of a gD chimericprotein of the present invention includes the entire gD amino acidsequence (e.g., amino acids 1-394) or the mature gD amino acid sequence(e.g., amino acids 26-394, the carboxy-terminal 369 amino acids). Inother embodiments, the first polypeptide segment includes less thanfull-length mature gD but includes 250, 260, 270, 280, 290, 300, 305,310, 315, 320, 325, 330, 335, 340, 345, 350, 351, 352, 353, 354, 355,356, 357, 358, 359, 360, 360, 361, 362, 363, 362, 365, 366, 367 or 368amino acids of the mature gD sequence.

In certain aspects, an antigenic amino acid sequence is inserted withina region of a gD that is between amino acids 230 and 300, between aminoacids 235 and 295, or between amino acids 240 and 290 of a mature gDamino acid sequence. In other aspects an antigenic amino acid sequenceis inserted at a position carboxy-terminal to amino acid 230, 235, 240,245, 250, 255, 260, 265, 270, 275, 280, 285 of a mature gD amino acidsequence. In certain aspects, an antigenic amino acid sequence isinserted immediately adjacent to amino acid 240, 241, 242, 243, 244,245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258,259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272,273, 274, 275, 276, 277, 278, 279, 280, 282, 282, 283, 284, 285, 286,287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, or 300of a mature gD amino acid sequence

In certain embodiments, a chimeric gD protein of the invention has astructure that is similar to the structure of the wild-type protein,that is, the chimeric gD protein has the ability to interact with HVEMand, in addition, may have one or more of the following activities: 1)stimulating a CD8⁺ T cell response to the fusion partner; 2) disruptingan HVEM-BTLA pathway activity; 3) interacting with nectin-1; 4)mediating cell entry by an HSV-1 and/or HSV-2 virus; and/or 5) mediatingcell-to-cell spread of HSV-1 and/or HSV-2.

Antigens

As used herein, the term “antigen” (also termed “fusion partner” inrelation to gD) is intended to include, but is not limited to, asubstance that an immune response is specifically mounted against, suchas a protein or a polypeptide. An antigen of the invention can be of anylength, ranging in size from a few amino acids in length to hundreds ofamino acids in length, provided that that the chimeric gD maintains astructure that is similar to that of the wild-type gD, e.g., thechimeric gD retains one or more activities of the wild-type gD,particularly the activities of interacting with HVEM and/or stimulatinga CD8⁺ T cell response to the fusion partner in the host. In certainaspects of the invention, the antigen is 1000, 900, 800, 700, 600, 500,400, 350, 340, 330, 320, 310, 300, 290, 280, 270, 260, 250, 240, 230,220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80,70, 60, 50, 40, 30, 20, 10 or less amino acids in length.

An antigen of the present invention includes a heterologous proteinand/or polypeptide, such as a viral, bacterial, fungal, or parasiteprotein or polypeptide, and/or a host polypeptide and/or protein e.g., atumor cell polypeptide or protein (such as an oncoprotein or a portionthereof) or a polypeptide or protein associated with inflammation.Antigens of particular interest include, but are not limited to,influenza virus antigens, such as nucleoprotein P (NP; see FIG. 16),matrixprotein (M), and hemagglutinin (HA); Plasmodium antigens such asthrombospondin-related anonymous protein (TRAP; see FIG. 17),ring-infected erythrocyte surface antigen (RESA), merozoite surfaceprotein 1 (MSP1), merozoite surface protein 2 (MSP2), merozoite surfaceprotein 3 (MSP3), and glutamate-rich antigen (GLURP); human papillomavirus (HPV) antigens, particularly HPV-16 antigens, such as E5 protein,E6 protein, and E7 protein; and HIV antigens, such as gag, pol, nef,tet, and env.

Viruses include DNA or RNA animal virus. As used herein, RNA virusesinclude, but are not limited to, virus families such as picoruaviridae(e.g., polioviruses), reoviridae (e.g., rotaviruses), logaviriclae(e.g., encephalitis viruses, yellow fever virus, rubella virus),orthomyxoviridae (e.g., influenza viruses), paramyxoviridae (e.g.,respiratory syncytial virus (RSV), measles virus (MV), mumps virus(MuV), parainfluenza virus (PIV)), rhabdoviridae (e.g., rabies virus(RV)), coronaviridae, bunyaviridae, flaviviridae (e.g., hepatitis Cvirus (HCV)), filoviridae, arenaviridae, bunyaviridae, and retroviridae(e.g., human T-cell lymphotropic viruses (HTLV), human immunodeficiencyviruses (HIV)). As used herein, DNA viruses include, but are not limitedto, virus families such as papovaviridae (e.g., papilloma viruses),adenoviridae (e.g., adenovirus), herpesviridae (e.g., herpes simplexviruses, e.g., HSV-1, HSV-2; varicella zoster virus (VZV); Epstein-Barrvirus (EBV); cytomegalovirus (CMV); human herpesviruses, e.g., HHV-6 andHHV-7; Kaposi's sarcoma-associated herpesvirus (KSHV) and the like), andpoxviridae (e.g., variola viruses). These and other viruses and viralproteins are included in the present invention and are described furtherin Knipe et al. Field's Virology, 4th ed., Lippincott Williams &Wilkins, 2001, incorporated herein by reference in its entirety for allpurposes.

Bacteria include, but are not limited to, gram positive bacteria, gramnegative bacteria, acid-fast bacteria and the like. As used herein, grampositive bacteria include, but are not limited to, Actinomedurae,Actinomyces israelii, Bacillus anthracis, Bacillus cereus, Clostridiumbotulinum, Clostridium difficile, Clostridium perfringens, Clostridiumtetani, Corynebacterium, Enterococcus faecalis, Listeria monocytogenes,Nocardia, Propionibacterium acnes, Staphylococcus aureus, Staphylococcusepiderm, Streptococcus mutans, Streptococcus pneumoniae and the like. Asused herein, gram negative bacteria include, but are not limited to,Afipia felis, Bacteriodes, Bartonella bacilliformis, Bortadellapertussis, Borrelia burgdorferi, Borrelia recurrentis, Brucella,Calymmatobacterium granulomatis, Campylobacter, Escherichia coli,Francisella tularensis, Gardnerella vaginalis, Haemophilias aegyptius,Haemophilias ducreyi, Haemophilias influenziae, Heliobacter pylori,Legionella pneumophila, Leptospira interrogans, Neisseria meningitidia,Porphyromonas gingivalis, Providencia sturti, Pseudomonas aeruginosa,Salmonella enteridis, Salmonella typhi, Serratia marcescens, Shigellaboydii, Streptobacillus moniliformis, Streptococcus pyogenes, Treponemapallidum, Vibrio cholerae, Yersinia enterocolitica, Yersinia pestis andthe like. As used herein, acid-fast bacteria include, but are notlimited to, Mycobacterium avium, Mycobacterium leprae, Mycobacteriumtuberculosis and the like.

Other bacteria not falling into the other three categories include, butare not limited to, Bartonella henselae, Chlamydia psitlaci, Chlamydiatrachomatis, Coxiella burnetii. Mycoplasma pneumoniae, Rickettsia akari,Rickettsia prowazekii, Rickettsia rickeltsii, Rickettsia tsutsugamushi,Rickettsia typhi, Ureaplasma urealyticum, Diplococcus pneumoniae,Ehrlichia chafensis, Enterococcus faecium, Meningococci and the like.

Fungi include, but are not limited to, Aspergilli, Candidae, Candidaalbicans, Coccidioides immitis, Cryptococci, and combinations thereof.

Parasites include, but are not limited to, Balantidium coli,Cryptosporidium parvum, Cyclospora cayatanensis, Encephalitozoa,Entamoeba histolytica, Enterocytozoon bieneusi, Giardia lamblia,Leishmaniae, Plasmodii, Toxoplasma gondii, Trupanosomae, trapezoidalamoeba and the like.

Oncoproteins are intended, without limitation, to refer to proteinsand/or peptides that are capable of inducing cell transformation.Oncoproteins include, but are not limited to, cellular proteins such asPDGF, ERB-B, ERB-B2, K-RAS, N-RAS, C-MYC, N-MYC, L-MYC, BCL-2, BCL-1,MDM2 and the like. Oncoproteins also include, but are not limited to,viral proteins from RNA and/or DNA tumor viruses such as hepatitis Bviruses, SV40 viruses, polyomaviruses, adenoviruses, herpes viruses,retroviruses and the like. Tumor suppressor proteins are intended,without limitation, to refer to proteins or polypeptides that cansuppress or block aberrant cellular proliferation, as well as tumorsuppressor proteins that have been mutated and, accordingly, no longersuppress or block aberrant cellular proliferation. Tumor suppressorproteins include, but are not limited to, cellular proteins such as APC,DPC4. NF-1, NF-2, MTS1, RB, p53 and the like.

gD chimeric proteins of the present invention are useful for modulatingdisorders associated with aberrant cellular proliferation mediated byoncoproteins and/or tumor suppressor proteins, such as cancer. Aberrantcellular proliferation is intended to include, but is not limited toinhibition of proliferation including rapid proliferation. As usedherein, the term “disorder associated with aberrant cellularproliferation” includes, but is not limited to, disorders characterizedby undesirable or inappropriate proliferation of one or more subset(s)of cells in a multicellular organism. The term “cancer” refers tovarious types of malignant neoplasms, most of which can invadesurrounding tissues, and may metastasize to different sites (PDR MedicalDictionary 1st edition (1995)). The terms “neoplasm” and “tumor” referto an abnormal tissue that grows by cellular proliferation more rapidlythan normal and continues to grow after the stimuli that initiatedproliferation is removed (PDR Medical Dictionary: 1st edition (1995)).Such abnormal tissue shows partial or complete lack of structuralorganization and functional coordination with the normal tissue whichmay be either benign (benign tumor) or malignant (malignant tumor).

Polypeptides and proteins associated with inflammation include thosethat modulate a disease or disorder characterized by, caused by,resulting from, or becoming affected by inflammation. Examples ofinflammatory diseases or disorders include, but not limited to, acuteand chronic inflammation disorders such as asthma, psoriasis, rheumatoidarthritis, osteoarthritis, psoriatic arthritis, inflammatory boweldisease (Crohn's disease, ulcerative colitis), sepsis, vasculitis, andbursitis; autoimmune diseases such as lupus, polymyalgia, rheumatica,scleroderma, Wegener's granulomatosis, temporal arteritis,cryoglobulinemia, and multiple sclerosis; transplant rejection;reperfusion injury in strokes or myocardial infarction; osteoporosis;cancer, including solid tumors (e.g., lung, CNS, colon, kidney, andpancreas); Alzheimer's disease; atherosclerosis; viral (e.g., HIV orinfluenza) infections; chronic viral (e.g. Epstein-Barr,cytomegalovirus, herpes simplex virus) infection; and ataxiatelangiectasia.

Nucleic Acid Molecules

The invention also provides nucleic acid molecules which encode fusionproteins of the invention. In certain embodiments of the invention, thenucleic acid molecule is a vector. As used herein, the term “vector”refers to a nucleic acid molecule, a protein, or a liquid structurecapable of transporting another nucleic acid. One type of vector is a“plasmid.” which refers to a circular double stranded DNA loop intowhich additional DNA segments can be ligated.

Another type of vector is a viral vector, wherein additional DNAsegments can be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors arereplication-defective and remain in the nucleus as episomes. Othervectors (e.g., non-episomal mammalian vectors) are integrated into thegenome of a host cell upon introduction into the host cell, and therebyare replicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “expressionvectors.” In general, expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids. The terms “plasmid” and“vector” can be used interchangeably as the plasmid is a commonly usedform of vector.

In one embodiment, a recombinant virus is provided for eliciting animmune response in a host infected by the virus. In certain aspects, therecombinant virus is replication-incompetent. A recombinant virus may beconstructed from any virus using methods known in the art, provided thatthe native progenitor is rendered replication incompetent. For example,replication-incompetent adenovirus, adeno-associated virus, SV40 virus,retrovirus, herpes simplex virus or vaccinia virus may be used togenerate the recombinant virus by inserting the viral antigen into aregion that is non-essential to the infectivity of the recombinantvirus. A recombinant virus does not have the pathologic regions of thenative progenitor of the benign virus but retains its infectivity to thehost.

In a certain embodiment, the recombinant virus is areplication-incompetent chimpanzee-derived adenovirus.Chimpanzee-derived adenovirus vectors have distinct advantages overpreviously used adenoviral recombinants of the human serotype 5 that istypically used in the art. Most importantly, the efficacy of simianadenoviral vaccine carriers is not impaired by pre-existing neutralizingantibodies to human adenovirus serotype 5 that can be detected in up to45% of the adult human population in the United States. Furthermore,simian adenoviral recombinants have interactions with cells of theinnate immune system, most notably dendritic cells, which sponsordevelopment of strongly biased Th1 responses suited to induce potentresponses of CD8⁺ T cells, a subset of immunocytes that is particularlyimportant to control the spread of HIV-1. For a review ofreplication-incompetent chimpanzee-derived adenovirus, see U.S. Pat. No.6,019,978, incorporated herein by reference in its entirety for allpurposes.

A number of viral vectors suitable for in vivo expression of the gDchimeric proteins described herein are known. Such vectors includeretroviral vectors (see, e.g., Miller (1992) Curr. Top. Microbiol.Immunol. 158:1; Salmons and Gunzburg (1993) Human Gene Therapy 4:129;Miller et al. (1994) Meth. Enz. 217:581) and adeno-associated vectors(reviewed in Carter (1992) Curr. Opinion Biotech. 3:533; Muzcyzka (1992)Curr. Top. Microbiol. Immunol. 158:97). Other viral vectors that areused include adenoviral vectors, alphavirus replicons, herpes virusvectors, pox virus vectors, and rhabdovirus vectors, as generallydescribed in, e.g. Jolly (1994) Cancer Gene Therapy 1:51; Latchman(1994) Molec. Biotechnol. 2:179; Johanning et al. (1995) Nucl. AcidsRes. 23:1495; Berencsi et al. (2001) J. Infect. Dis. 183:1171;Rosenwirth et al. (2001) Vaccine February 19:1661; Kittlesen et al.(2000) J. Immunol. 164:4204; Brown et al. (2000) Gene Ther. 7:1680;Kanesa-thasan et al. (2000) Vaccine 19:483; and Sten (2000) Drug 60:249.Compositions comprising vectors and an acceptable excipient are providedherein.

Nucleic Acid and Protein Variants

In certain aspects, gD nucleic acid molecules and polypeptides are“naturally occurring.” As used herein, a “naturally-occurring” moleculerefers to a gD molecule having a nucleotide sequence that occurs innature (e.g., encodes a gD polypeptide sequence found in a herpessimplex virus, e.g., HSV-1 or HSV-2). In addition, naturally ornon-naturally occurring variants of these polypeptides and nucleic acidmolecules which retain the same functional activity, e.g. the ability tobind HVEM and or stimulate an immune response to a fusion partner in ahost. Such variants can be made, e.g., by mutation using techniques thatare known in the art. Alternatively, variants can be chemicallysynthesized.

As used herein, the term “variant” is intended to include, but is notlimited to, nucleic acid molecules or polypeptides that differ insequence from a reference nucleic acid molecule or polypeptide, butretains its essential properties, that is, it retains the ability tointeract with HVEM and, in addition, it may have one or more of thefollowing activities: 1) stimulating a CD8⁺ T cell response to a fusionpartner 2) disrupting an HVEM-BTLA pathway activity; 3) interacting withnectin-1; 4) mediating cell entry by an HSV-1 and/or HSV-2 virus; and/or5) mediating cell-to-cell spread of HSV-1 and/or HSV-2. Changes in thenucleotide sequence of the variant may or may not alter the amino acidsequence of a polypeptide encoded by the reference nucleic acidmolecule. Nucleotide changes may result in amino acid substitutions,additions, deletions, fusions and truncations in the polypeptide encodedby the reference sequence.

Variants can be made using mutagenesis techniques that are known in theart. Alternatively, variants can be chemically synthesized. Mutationscan include one or more point mutations, deletions and/or insertions. Incertain aspects of the invention, a mutant gD chimeric polypeptide hasthe ability to bind HVEM at a level that is the same as or greater thanthe ability of a wild-type gD protein to bind HVEM. In certain aspectsof the invention, amino acid W 294 of the mature gD sequence is mutatedto alanine.

Construction of Fusion Proteins

Fusion proteins of the invention typically are prepared recombinantly,as described in Example 1. Many kits for constructing fusion proteinsare available from companies such as Promega Corporation (Madison,Wis.), Stratagene (La Jolla, Calif.), CLONTECH (Mountain View, Calif.),Santa Cruz Biotechnology (Santa Cruz, Calif.), MBL InternationalCorporation (MIC; Watertown, Mass.), and Quantum Biotechnologies(Montreal, Canada; 1-888-DNA-KITS). Alternatively, a fusion protein canbe synthesized chemically, for example, using solid phase techniques.See, e.g., Merrifield, J. Am. Chem. Soc. 85, 2149 54, 1963; Roberge etal. Science 269, 202 04, 1995. Protein synthesis can be performed usingmanual techniques or by automation. Automated synthesis can be achieved,for example, using Applied Biosystems 431A Peptide Synthesizer (PerkinElmer). Optionally, fragments of a fusion protein can be separatelysynthesized and combined using chemical methods to produce a full-lengthmolecule.

Methods of Using Fusion Proteins and Nucleic Acids of the Invention

Pharmaceutical Compositions

Dosage Regimens

Certain embodiments of the invention are directed to prophylacticallytreating an individual in need thereof. As used herein, the term“prophylactic treatment” includes, but is not limited to, theadministration of a nucleic acid sequence encoding a gD chimeric proteinto a subject who does not display signs or symptoms of a disease,pathology, or medical disorder, or displays only early signs or symptomsof a disease, pathology, or disorder, such that treatment isadministered for the purpose of diminishing, preventing, or decreasingthe risk of developing the disease, pathology, or medical disorder. Aprophylactic treatment functions as a preventative treatment against adisease or disorder.

Certain embodiments of the invention are directed to therapeuticallytreating an individual in need thereof. As used herein, the term“therapeutically” includes, but is not limited to, the administration ofa nucleic acid sequence encoding a gD chimeric protein to a subject whodisplays symptoms or signs of pathology, disease, or disorder, in whichtreatment is administered to the subject for the purpose of diminishingor eliminating those signs or symptoms of pathology, disease, ordisorder.

Embodiments of the present invention are directed to compositions andmethods for enhancing the immune response of a subject to one or moreantigens. As used herein, the terms “subject” and “host” are intended toinclude living organisms such as mammals. Examples of subjects or hostsinclude, but are not limited to, horses, cows, sheep, pigs, goats, dogs,cats, rabbits, guinea pigs, rats, mice, gerbils, non-human primates,humans and the like, non-mammals, including, e.g., non-mammalianvertebrates, such as birds (e.g., chickens or ducks) fish or frogs(e.g., Xenopus), and a non-mammalian invertebrates, as well astransgenic species thereof.

As used herein, the term “immune response” is intended to include, butis not limited to, T and/or B cell responses, that is, cellular and/orhumoral immune responses. In one embodiment, the claimed methods can beused to stimulate cytotoxic T cell responses. The claimed methods can beused to stimulate both primary and secondary immune responses. Theimmune response of a subject can be determined by, for example, assayingantibody production, immune cell proliferation, the release ofcytokines, the expression of cell surface markers, cytotoxicity, and thelike. In certain aspects, the claimed gD chimeric proteins increase theimmune response in a subject when compared to the immune response by anuntreated subject or a subject who receives a vaccine containing thesame antigen but without gD.

As used herein, the term “enhancing an immune response” includesincreasing T and/or B cell responses, that is, cellular and/or humoralimmune responses, by treatment of a subject using the claimed gDchimeric proteins and/or methods. In one embodiment, the claimed gDchimeric proteins and/or methods can be used to enhance cytotoxic T cellresponses to the antigen (fusion partner). In another embodiment, theclaimed compounds and methods can be used to inhibit the ability of theHVEM to interact with B and T lymphocyte attenuator (BTLA), thusenhancing an immune response to the antigen (fusion partner). In anotherembodiment, the claimed gD chimeric protein interacts with HVEM.

As used herein, the term “immune cell” is intended to include, but isnot limited to, cells that are of hematopoietic origin and play a rolein an immune response. Immune cells include, but are not limited to,lymphocytes, such as B cells and T cells; natural killer cells: myeloidcells, such as monocytes, macrophages, eosinophils, mast cells,basophils, and granulocytes.

As used herein, the term “adjuvant” includes, but is not limited to,agents which potentiate the immune response to an antigen. Adjuvants canbe administered in conjunction with a nucleic acid sequence encoding agD chimeric protein of the invention to additionally augment the immuneresponse.

Nucleic acid sequences encoding the gD chimeric proteins describedherein can be administered to subjects in whom it is desirable topromote an immune response. In one embodiment, a nucleic acid sequenceencoding a gD chimeric protein described herein is administeredprophylactically, e.g., prior to infection with a pathogen or to asubject who is free of cancer or free of an autoimmune disease. Inanother embodiment, a nucleic acid sequence encoding a gD chimericprotein described herein is administered therapeutically, e.g., to asubjects who has a preexisting condition, e.g., a subject who isinfected with a pathogen, who has cancer, or who suffers from anautoimmune disease.

In one embodiment, the gD chimeric protein is administered by “geneticimmunization.” In this embodiment, a DNA expression vector encoding thegD chimeric protein is injected into the subject animal, e.g., into theskin or into a muscle of the subject. The gene products are correctlysynthesized, glycosylated, folded, and expressed by the subject toelicit the desired immune response. In one embodiment, DNA is injectedinto muscles or delivered into the skin coated onto gold microparticlesby a particle bombardment device, a “gene gun.” Genetic immunization hasbeen shown to induce specific humoral responses and cellular immuneresponses (See, e.g., Mor et al. (1995) J. Immunol. 155:2039; Xu andLiew (1995) Immunology 84:173; Davis et al. (1994) Vaccine 12:1503).

A dosage regimen of administration of a gD chimeric protein or a nucleicacid sequence encoding a gD chimeric protein may be adjusted to providethe optimum therapeutic response for each subject without undueexperimentation. For example, antibody titers to an antigen or cellularimmune responses to an antigen can be measured to determine whether ornot the subject is developing an immune response or is manifesting anenhanced immune response to the antigen and the dosage regimen can beadjusted accordingly.

The composition including a gD chimeric protein or a nucleic acidsequence encoding a gD chimeric protein may also be administeredparenterally or intraperitoneally. The agent can be administered, forexample, intranasally, orally, intravenously, intramuscularly,subcutaneously or mucosally. Dispersions can also be prepared inglycerol, liquid polyethylene glycols, and mixtures thereof and in oils.Under ordinary conditions of storage and use, these preparations maycontain a preservative to prevent the growth of microorganisms.

Pharmaceutical compositions suitable for injection include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. A pharmaceutical composition of the inventioncan be formulated to be suitable for a particular route ofadministration. For example, in various embodiments, a pharmaceuticalcomposition of the invention can be suitable for injection, inhalationor insufflation (either through the mouth or the nose), or forintranasal, mucosal, oral, buccal, parenteral, rectal, intramuscular,intravenous, intraperitoneal, and subcutaneous delivery.

The composition including a gD chimeric protein or a nucleic acidsequence encoding a gD chimeric protein will be sterile. In addition, itwill be stable under the conditions of manufacture and storage andpreserved against the contaminating action of microorganisms such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, and liquid polyethylene glycol, and the like), andsuitable mixtures thereof. The proper fluidity can be maintained, forexample, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms can be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be desirable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile, injectable solutions can be prepared by incorporating a gDchimeric protein or a nucleic acid sequence encoding a gD chimericprotein in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfilter sterilization. Generally, dispersions are prepared byincorporating the gD chimeric protein or the nucleic acid sequence (withor without a carrier) encoding the gD chimeric protein into a sterilevehicle which contains a basic dispersion medium and the required otheringredients from those enumerated above. In the case of sterile powdersfor the preparation of sterile injectable solutions, the preferredmethods of preparation are vacuum drying and freeze-drying which yieldsa powder of the active ingredient (e.g., agent or composition) plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof. The agent or composition can be administered in a formsuitable for use with a needle-less injector device (such devices areknown in the art (see, e.g., U.S. Pat. Nos. 5,383,851; 5,581,198;5,846,233) for example as described in Mol. Med. (1998) 4:109.

When the composition including a gD chimeric protein or a nucleic acidsequence encoding a gD chimeric protein is suitably protected, asdescribed above, the composition may be orally administered, forexample, with an inert diluent or an assimilable edible carrier. As usedherein “pharmaceutically acceptable carrier” includes any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like. The useof such media and agents for pharmaceutically active substances is wellknown in the art. Except insofar as any conventional media or agent isincompatible with the composition including a gD chimeric protein or anucleic acid sequence encoding a gD chimeric protein, use thereof in thetherapeutic compositions is contemplated. Supplementary active compoundscan also be incorporated into the compositions.

It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the mammalian subjects to be treated; eachunit containing a predetermined quantity of active compound calculatedto produce the desired therapeutic effect in association with therequired pharmaceutical carrier. The specification for the dosage unitforms of the invention are dictated by and directly dependent on (a) theunique characteristics of the active compound and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an active agent or composition for thetreatment of individuals.

The composition including a gD chimeric protein or a nucleic acidsequence encoding a gD chimeric protein of the invention is administeredto subjects in a biologically compatible form suitable forpharmaceutical administration in vivo to enhance immune responses. By“biologically compatible form suitable for administration in vivo” ismeant a form of the gD chimeric protein or a nucleic acid sequenceencoding a gD chimeric protein to be administered in which any toxiceffects are outweighed by the therapeutic effects of the agent.

Administration of a therapeutically or prophylactically active amount ofthe compositions of a gD chimeric protein or a nucleic acid sequenceencoding a gD chimeric protein is defined as an amount effective, atdosages and for periods of time necessary to achieve the desired result.The administration of a gD chimeric protein or a nucleic acid sequenceencoding a gD chimeric protein can result in an enhanced immune response(e.g., a stimulation of CD8⁺ T cells) to an antigen (e.g., a viral or atumor cell antigen).

As defined herein, a therapeutically or prophylactically effectiveamount of a composition of a gD chimeric protein or a nucleic acidsequence encoding a gD chimeric protein (an effective dosage) rangesfrom about 0.001 to 30 mg/kg body weight, about 0.01 to 25 mg/kg bodyweight, about 0.1 to 20 mg/kg body weight, or from about 1 to 10 mg/kg,2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight.The skilled artisan will appreciate that certain factors may influencethe dosage required to effectively treat a subject, including but notlimited to the severity of the disease or disorder, previous treatments,the general health and/or age of the subject, and other diseasespresent. Moreover, treatment of a subject with a therapeuticallyeffective amount of an inhibitor can include a single treatment or caninclude a series of treatments. It will also be appreciated that theeffective dosage of inhibitor used for treatment may increase ordecrease over the course of a particular treatment. Changes in dosagemay result from the results of diagnostic assays as described herein.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology, microbiology, recombinant DNA and immunology, whichare within the skill of the art. Such techniques are explained fully inthe literature. See, for example, Molecular Cloning A Laboratory Manual,2nd Ed., ed. by Sambrook, J. et al. (Cold Spring Harbor Laboratory-Press(1989)); Short Protocols in Molecular Biology, 3rd Ed., ed. by Ausubel,F. et al. (Wiley, NY (1995)); Current Protocols in Molecular Biology,John Wiley & Sons, Inc., 1998; DNA Cloning, Volumes I and II (D. N.Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed. (1984));Mullis et al. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B. D.Hames & S. J. Higgins eds. (1984)); the treatise, Methods In Enzymology(Academic Press, Inc., N.Y.); Immunochemical Methods in Cell andMolecular Biology (Mayer and Walker, eds., Academic Press, London(1987)); Handbook of Experimental Immunology, Volumes I IV (D. M. Weirand C. C. Blackwell, eds. (1986)); and Miller, J. Experiments inMolecular Genetics (Cold Spring Harbor Press, Cold Spring Harbor, N.Y.(1972)).

The following examples are set forth as being representative of thepresent invention. These examples are not to be construed as limitingthe scope of the invention as these and other equivalent embodimentswill be apparent in view of the present disclosure, figures, tables, andaccompanying claims. The contents of all references, patents andpublished patent applications cited throughout this application arehereby incorporated by reference in their entirety for all purposes.

Example 1 Materials and Methods

Consultation of HSV-1 gD Fused Genes

A number of DNA and adenovirus vector vaccines were constructed andtested (Table 1). The chimeric gene gDE7E6E5 was constructed based onthe fusion of the HPV-16 E7, E6 and E5 oncoproteins and the HSV-1 gDprotein. Although in this example the HPV proteins are in the order E7,E6, and E5, they can be used in any order. The E7, E6 and E5 genes,without their respective stop codons, were amplified by PCR using theHPV-16 complete genome as a template. The gD gene is from HSV-1. E7, E6,and E5 genes are from HPV-16. Gag is a codon-optimized truncated form ofgag from HIV-1 clade B. SgD is a mutated form (W294A) of HSV-1 gD, whichshows high affinity to HVEM (Krummenacher et al., 2005). NBEF is amutated HSV-1 gD, which contains mutations that has been described toprevent gD-HVEM interaction (Connelly et al, 2003); see SEQ ID NO:37.pRE4 was provide by Drs. Gary Cohen and Roselyn Eisenberg (Cohen et al.,1988). AdC68gag was previously described by Fitzgerald and collaborators(2003).

TABLE 1 List of vaccine vectors used. Vector name Genes encoded Vaccinecarrier pRE4 (pgD) gD DNA vaccine pE7E6E5 E7, E6, and E5 DNA vaccinepgDE7E6E5 gD, E7, E6, and E5 DNA vaccine pgDE7 gD and E7 DNA vaccinepSgDE7 SgD (gDW294A) and E7 DNA vaccine pSgDE7E6E5 SgD, E7, E6, and E5DNA vaccine pNBEFgDE7E6E5 NBEFgD, E7, E6 and E5 DNA vaccine pgag gag DNAvaccine pgDgag gD and gag DNA vaccine AdC68gD gD E1-deleted adenovirusvector, chimpanzee serotype 68 AdC68E7 E7 E1-deleted adenovirus vector,chimpanzee serotype 68 AdC68E7E6E5 E7, E6, and E5 E1-deleted adenovirusvector, chimpanzee serotype 68 AdC68gDE7E6E5 gD, E7, E6, and E5E1-deleted adenovirus vector, chimpanzee serotype 68 AdC68gag gagE1-deleted adenovirus vector, chimpanzee serotype 68 AdC68gDgag gD andgag E1-deleted adenovirus vector, chimpanzee serotype 68

Separate amplification reactions were carried out with the followingprimers: E7FwApaI and E7RvNarI, E6FwNarI and E6RvNotI, and E5FwNotI andE5RvApaI (Table 2). The DNA fragment of the E7 gene was cleaved withApaI and NotI. The E6 DNA fragment was cleaved with NotI and NarI, andthe E5 was cleaved with NarI and ApaI. All DNA fragments were clonedinto the ApaI site in the pRE4 vector, provided by Drs. Gary Cohen andRoselyn Eisenberg (University of Pennsylvania, USA) (Cohen et al.,1988). The correct in-frame cloning of E7-, E6- and E5-encoding geneswas confirmed after nucleotide sequencing at Wistar Sequencing Facility.Control vectors pE7E6E5 and AdC68E7E6E5 were generated by PCR usingpgDE7E6E5 as template and primers E7FwHindIII and ESRvHindIII (Table 2).AdC68gD control vector was generated using pRE4 as template and primersgDFwXbaI and gDRvXbaI (Table 2).

TABLE 2 List of primers used Primer Sequence (5′-3′) E7FwApaIGCTGTAGGGCCCCATGGAGATACACCTAC (SEQ ID NO: 1) E7RvNarICATGGTGGCGCCTGGTTTCTGAGAACAG (SEQ ID NO: 2) E6FwNarIAGACATGGCGCCCACCAAAAGAGAACTGC (SEQ ID NO: 3) E6RvNotICTCCATGCGGCCGCCCAGCTGGGTTTCTCTACG (SEQ ID NO: 4) E5FwNotIGACAAAGCGGCCGCCTGCATCCACAACATTAC (SEQ ID NO: 5) E5RvApaIACATATGGGCCCTGTAATTAAAAAGCGTGC (SEQ ID NO: 6) E7FwHindIIIGGGTGGAAGCTTATGGGAGATACACCTAC (SEQ ID NO: 7) E5RvHindIIITGGGGCAAGCTTTTAAATTAAAAAGCGTGC (SEQ ID NO: 8) gdFwXbaICCCTAGTCTAGAATGGGGGGGGCTGCCGCC (SEQ ID NO: 9) gDRvXbaICCCTAGTCTAGACTAGTAAAACAAGGGCTGGTG (SEQ ID NO: 10) gagFwNarIAAGAAGGGCGCCGGTGCGAGAGCGTCAG (SEQ ID NO: 11) gagRvNarIAAGGGTGGCGCCCAAAACTCTTGCCTTATGGC (SEQ ID NO: 12) gDFwHindIIIAAGCCCAAGCTTATGGGGGGGGCTGCCGCC (SEQ ID NO: 13) gDRvHindIIIAAGCCCAAGCTTCTAGTAAAACAAGGGCTGGTG (SEQ ID NO: 14) NBEFgDRvGACCGGAAGGTCTTTGCCGCGAAAGCGAGCGGGGTCGGCCGCCTTGAG (SEQ ID NO:15) NBEFgDFwCGCTTTCGCGGCAAAGACCTTCCGGTCGCGGACGCGGCGGCCGCCCC (SEQ ID NO:16) SgDFwCAAATCCAACAAAACGCGCACATAGGCTCGATCC (SEQ ID NO: 17) SgDRvGATCGACGGTATGTGCGCGTTTGGTGGGATTTGC (SEQ ID NO: 18)

To construct the AdC68gDE7E6E5 vector, the gDE7E6E5 chimeric gene wasamplified by PCR using the pgDE7E6E5 vector as a template. The PCRreaction was carried out with gDFwXbaI and gDRvXbaI primers (Table 2).The DNA fragment of the gDE7E6E5 chimeric gene was cleaved with XbaI andcloned into XbaI site on the shuttle vector (BD PharMingen. San Diego.CA). The pShuttlegDE7E6E5 clone was confirmed by restriction analysisand sub-cloned into E1-deleted chimpanzee-derived adenovirus vectorserotype 68 using PI-SceI and I-CenI sites as described (Fitzgerald etal. 2003).

The gDgag chimeric gene was generated by insertion of thecodon-optimized truncated form of gag from HIV-1 clade B into the HSV-1gD NarI site. The gag gene was amplified by PCR using the pCMVgag vectoras a template and primers gagFwNarI and gagRvNarI (Table 2). The DNAfragment corresponding to gag gene was cleaved with NarI, cloned intopShuttlegD, and then sub-cloned into AdC68 vector as described above.

Construction of gD Mutants

The SgDE7 mutated gene was constructed using QUICKCHANGE® site-directedmutagenesis kit (Stratagene Cloning Systems, La Jolla, Calif.) asrecommended by the manufacturer. Briefly, SgDFw and SgDRv primers (Table2), designed to mutate the amino acid residue 294 of gD, were used toPCR amplify the entire pgDE7 vector. The reaction products were thentreated with DpnI and used to transform DH5α E. coli cells. TheNBEFgDF7E6E5 gene (see SEQ ID NO:37) was generated by mutation ofresidues crucial for HVEM-gD interaction. HSV-1 gD residues 11, 15, 25,27, 28, 29, and 30, were mutated to alanine by gene splicing by overlapextension (i.e., gene SOEing). Briefly, two PCR reactions were carriedout using set of primers, (i) with gDFwHindIII and NBEFgDRv, and (ii)with NBEFgDFw and gDRvHindIII (Table 2). Vector pgDE7E6E5 was used as atemplate in both PCR reactions. Two amplified fragments were used astemplate to PCR reaction with gDfWHindIII and gDRvHindIII primers (Table2). The NBEFgDE7E6E5 DNA fragment was cloned into the same pgDE7E6E5backbone vector. Both mutant gD sequences were confirmed by sequencingthe entire gene at Wistar Sequencing Facility.

DNA Vaccine and E1-Deleted Chimpanzee-Derived Adenovirus Purification

DNA vaccines were propagated in E. coli K12 DH5α cells in LB mediumsupplemented with ampicillin and purified with the Maxi Prep Kit(QIAGEN®, Valencia, Calif.). The DNA concentration was determined byspectrophotometry at 260 nm and confirmed by visual inspection ofethidium bromide-stained 1% agarose gels in comparison to DNA fragmentsof known concentration (Invitrogen, Carlsbad, Calif.). Plasmids werekept at −20° C. until use, at which time the DNA concentration wasadjusted to 1 μg/μl in phosphate-buffered saline (PBS). AdC68 vectorswere propagated using E1-transfected HEK 293 cells and purified by CsClgradient centrifugation as previously described (Fitzgerald et al.,2003). Upon purification, the concentration of each virus vector batchwas determined by measuring virus particles (vp) by spectrophotometry at260 nm.

Cell Lines

TC-1 tumor cells, derived from C57BL/6 origin lung epithelial cellstransformed with v-Ha-ras and HPV-16 E6 and E7 genes, were provided byDr. T. C. Wu, Johns Hopkins University, USA (Lin et al., 1996). Mousemelanoma cells B78H1 and B78H1/3E5, which express HVEM fused to EGFP,were provided by Drs. Gary Cohen and Roselyn Eisenberg (University ofPennsylvania, USA). E1-transfected HEK 293 cells were used to propagateE1-deleted chimpanzee derived adenovirus vectors. All cells werepropagated in DMEM supplemented with glutamine, sodium pyruvate,nonessential amino acids, HEPES buffer, antibiotic, and 10% FBS (TC-1,CHO/CAR and E1-transfected HEK 293 cells) or 5% FBS (B78H1 and B78H1/3E5cells). CHO cells stably transfected to express the coxsackie adenovirusreceptor (CHO/CAR) were obtained from J. Bergelson (Childrens Hospitalof Philadelphia).

Animals and Immunization

Female BALB/c and C57Bl/6 mice at 6-8 weeks of age were purchased fromJackson Laboratory (Bar Harbor, Me.) or Charles River Laboratories(Boston, Mass.) and housed at the Animal Facility of the WistarInstitute. All procedures involving handling and sacrifice of animalswere performed according to approved protocols in accordance withrecommendations for the proper use and care of laboratory animals at theWistar Institute. Groups of 5 to 10 BALB/c and C57Bl/6 mice wereintramuscularly (i.m.) vaccinated with the DNA vaccines or E1-deletedchimpanzee derived adenovirus vectors into the tibialis anterior muscleof each hind limb. DNA vaccine was given at 100 μg divided in two 50 μlaliquots. E1-deleted chimpanzee-derived adenovirus vectors (AdC68) wereinoculated from 1×10⁸ to at 5×10¹⁰ vp per mouse. For most experimentsAdC68 vectors were inoculated at 10¹⁰ viral particles per mouse.

E7 Transgenic Mouse

The E7 transgenic mouse was based on a similar mouse where E7 isexpressed in the thymus under the control of the thyroglobulin promoter(Ledent et al., Oncogene 1995; 10:1789-97). The plasmid used to createthe E7 mouse was a very generous gift from Dr. Catherine Ledent,UniversitéLibre de Bruxelles. The plasmid, constructed in the pSG5vector, contained the bovine thyroglobulin promoter, a rabbit β-intron,the E7 gene, and a polyadenylation signal in a gene cassette. The bovinethyroglobulin promoter was used as it has been shown to be tightlyregulated and expressed in thyrocytes (Ledent et al., Proc Natl Acad SciUSA 87:6176, 1990). The rabbit β-intron was used to increase theexpression of the transgenes (Palmiter et al., Proc Natl Acad Sci USA A88:478, 1991). This cassette was removed and purified by gelelectrophoresis followed by a Geneclean kit (Q-Biogen, Morgan Irvine.CA). DNA was concentrated by ethanol precipitation. The cut DNA was thenmicroinjected by the University of Pennsylvania School of MedicineTransgenic Facility under the direction of Dr. Jean Richa. The foundermouse strain was C57BL/6. The founder mice were thus mated to wild typeC57BL/6, and the progeny back crossed and screened by the ACT real timePCR method for homozygosity. The E7 tg mice were bred at the AnimalFacility of the Wistar Institute from a pair provided by Dr. Y.Patterson (University of Pennsylvania). All animal procedures wereperformed in accordance with recommendations for the proper use and careof laboratory animals at the Wistar Institute. Groups of 3 to 10 animalswere vaccinated i.m. with the E1-deleted AdC68 vectors into the tibialisanterior muscle of each hind limb. AdC68 vectors were inoculated at5×10¹⁰ vp per mouse unless stated otherwise.

Intracellular Cytokine Staining

Intracellular IFN-γ staining was performed using peripheral bloodmononuclear cells (PBMC) and cells from the spleen two weeks after theDNA vaccine dose or 10 days after E1-deleted chimpanzee-derivedadenoviral vector administration unless stated otherwise. Samples werewashed twice with L-15 medium. Cells were then treated for 5 minutes onice with ACK lysis buffer (Invitrogen) to rupture red blood cells,washed, and suspended in DMEM supplement with 2% FBS. Samples werecultured at a concentration of 10⁶ cells/well for 5 hours at 37° C. in a96-well round bottom microtiter plate (Costar) in 200 μl of DMEMsupplemented with 2% FBS and 10⁻⁶ M 2-mercaptoethanol. Brefeldin A(GolgiPlug; BD PharMingen) was added at 1 μl/ml. The E7-specificRAHYNIVTF (SEQ ID NO: 19) peptide, which carries the immunodominantepitope of E7 for mice of the H-2b haplotype, or the AMQMLKETI (SEQ IDNO:20) peptide, which carries the immunodominant MHC class I epitope ofgag for mice of the H-2d haplotype, were used for peptide stimulation ata concentration of 3 μg/ml. The V3 control peptide delineated from thesequence of the envelope protein of HIV-1 clade B (VVEDEGCTNLSGF; SEQ IDNO:21) and the SIINFEKL peptide (SEQ ID NO:30) were used as controlpeptides. After washing, cells were incubated for 30 min at 4° C. with100 μl of a 1:100 dilution of a fluorescein (FITC)-conjugated monoclonalantibody to mouse CD8a (BD PharMingen). Cells were washed once with PBSfollowed by permeabilization with Cytofix/Cytoperm (BD PharMingen) for20 min at 4° C., washed twice with Perm/Wash buffer (BD PharMingen) andincubated in the same buffer for 30 min at 4° C. with 50 μl of a 1:100dilution of a phycoerythrin (PE)-labeled monoclonal antibody to mouseIFN-γ (BD PharMingen). After washing, cells were suspended in PBS andexamined by two-color flow cytometry using an EPICS Elite XL (BeckmanCoulter). Data were analyzed by WinMDi software. The percentages ofantigen specific CD8⁺ T cells that stained positive for IFN-γ over allCD8⁺ T cells were determined.

TC-1 Challenge

C57Bl/6 Mice were challenged subcutaneously (s.c.) with 1×10⁵ TC-1 cellssuspended in 100 μl of serum-free media, and injected at one rear flank.To determine the protection of pre-challenge vaccination mice werechallenged 10 and 14 days after vaccination with DNA vaccine orE1-deleted chimpanzee derived adenovirus vector, respectively.Post-challenge vaccination was evaluated with mice challenged five daysbefore vaccination (Example 3 and 15). Tumor growth in pre- andpost-challenge vaccinated mice was monitored by visual inspection andpalpation three times a week. Animals were scored as tumor-bearing whentumors attained sizes of approximately 1-2 mm in diameter. Mice wereeuthanized once tumors exceeded a diameter of 1 cm. Tumor growth wasfollowed for a period of 60 days after the challenge.

Statistical Analysis

Experiments were conducted using 3-10 mice per group. Samples tested byELISA were assayed in triplicates. Results show the means±standarddeviation (SD). Intracellular cytokine staining was conducted with PBMCfrom individual mice, while tetramer and markers staining were performedwith pooled samples. Significances between two groups were analyzed byone-tailed student's t-test.

HVEM Binding Assay

CHO/CAR cells were infected with either AdC68gD, AdC68gag, orAdC68gDgag. After 72 hrs, cells were harvested, suspend in 1 ml ofextraction buffer (10 mM Tris, 150 mM NaCl, 10 mM EDTA. 1% NP-40. 0.5%sodium deoxycholate, 1 mM PMSF [pH 8.0]) supplement with CompleteProtease inhibitor (Roche, Basel. Switzerland), and incubated at 4° C.for 1 hour. After spinning at 12,000 g for 15 min at 4° C., proteinextracts were kept at −80° C. until use. A capture enzyme-linkedimmunosorbent assay (ELISA) was used to normalize the amount of gD inthe extracts. ELISA plates were coated with 50 μl of a 10 μg/mlconcentration of ID3 monoclonal antibody (MAb) diluted in PBS/well.After an overnight incubation at 4° C., plates were exposed to blockingsolution for 1 hour and then to extracts diluted in blocking solutionfor 2 hours at room temperature. Captured gD was detected by adding 50μl of a 1 μg/ml concentration of Pab R7/well followed by goatanti-rabbit antibody coupled to horseradish peroxidase. Plates wererinsed with 20 mM citrate buffer (pH 4.5), ABTS peroxidase substrate wasadded, and the absorbance at 405 nm was recorded by using a microtiterplate reader. The level of gD in each extract was normalized by dilutionin extraction buffer. To assess receptor binding of the gD mutants,ELISA plates were coated overnight with 50 μl of human-HVEM (5 μg/ml),exposed to blocking solution, and incubated with normalized cellextracts diluted in blocking solution for 2 hours at room temperature.Bound gD was detected as described above, soluble gD306 (Nicola et al.,J. Virol. 71, 2940-46, 1997; Sisk et al., J. Virol. 6H, 766-75, 1994)and gD285 (Whitbeck et al. J. Virol. 71, 6083-93, 1997), purified asdescribed previously, were used at 1 μM.

Detection of Transcripts by Real-Lime PCR

Total RNA was isolated from CHO/CAR cells 48 hrs after infection withthe Ultraspec RNA solution system kit (Biotex). The mRNA was reversedtranscribed in vitro using MEGAscript transcription kit (Ambion).Remaining DNA was removed by treatment with DNase 1 (Ambion) for 1 h at37° C. Quantification of the housekeeping geneglyceraldehyde-3-phosphate dehydrogenase (GAPDH) was performed tonormalize the amount of cDNA in each sample. Normalized cDNA sampleswere used for amplification with E5-, E6-, E7- and gD-specific primers.Quantitative real-time PCR reactions were conducted using theLightCycler-RNA amplification kit SYBR Green 1 (Boehinger Mannheim),following manufacture's conditions. Samples were quantified intriplicate by three independent experiments.

Confocal Microscopy and FACS Analysis

B78H1/3E5 cells, which express HVEM fused to Enhanced Green FluorescenceProtein (HVEM-EGFP), or B78H1 cells (both obtained from Drs. Gary Cohenand Roselyn Eisenberg, University of Pennsylvania) were infected witheither AdC68gD, AdC68gag, or AdC68gDgag. CHO/CAR cells were infectedwith AdC68gD, AdC68E7E6E5, or AdC68gD-E7E6E5. After 48 hours, cells weredirectly stained or permeabilized with Cytofix/Cytoperm (BD PharMingen)and then stained with anti-gD MAb (DL-6) followed by anti-mouse IgGconjugated with Texas Red or PE. Confocal microscopy was performed witha Leica TCS SP2 Confocal Microscope at 400× final magnification. Cellsuspensions were analyzed using an EPICS XL (Beckman-Coulter, Inc.Miami, Fla.) to determine presence of gDgag. Data were analyzed byFlowjo software (Tree Star, Inc.).

Molecular Modeling of gD-gag

The 3-dimensional models of the gD-gag were constructed with theMODELLER package^(44,45) by combining the structures of individualprotein domains as determined by X-ray crystallography. The receptorbound form of gD-gag model was based upon the HSV-1 gD HVEM complex(1JMA)¹², chain A, residues 1:259; SIV gag (1ECW)⁴⁶, residues 1:119; andHIV-1 gag (1E6J)⁴⁷, chain P, residues 11:220. The gD-gag unligated formwas based upon the Cyclophilin A/HIV-1Chimera Complex (1M9D)⁴⁸, chain A,residues 1:15; HSV-1 gD (2C36)¹⁵, chain A, residues 23:256; SIV gag(1ECW), residues 1:119; and HIV-1 gag (1E6J), chain P, residues 11:220.Ribbon representations were prepared within the Swiss-PdbViewerprogram⁴⁹ and rendered with the Persistence of Vision Ray Tracer program(POV-Ray 2004, version 3.6).

In Vitro T Cell Proliferation Assay

Cells were harvested from draining popliteal lymph node of naïve andmice i.m. immunized 24 hrs earlier with 10¹¹ vps of AdC68gD orAdC68E7E6E5 then irradiated with 2000 RADs. CD8 cells were isolated fromthe spleens of OT-1 mice by negative selection using magnetic beads(Miltenyi Biotec) and labeled with 2 μM CFSE (Molecular Probes). A totalof 1×10⁶ irradiated lymph node cells were cultured with 1×10⁵ CD8⁺CFSE-labeled OT-1 cells in presence of either SIINFEKL peptide orcontrol peptide AMQMLKETI (both at 10⁻⁸ M, Alpha DiagnosticInternational) in 96-well plate wells for 72 hours. Cells were stainedwith anti-CD8-PerCP and anti-Vα2-PE (both BD Pharmingen) for 30 min onice. Cells were examined on aFACSCalibur using CellQuest software (BDBiosciences Pharmingen) and were analyzed using FlowJo software version7.1.2 (TriStar, Inc).

ELISA for Antibodies to gag

Sera from the vaccinated or naïve mice were tested on plates coated withpurified gag protein. Briefly, 96-well round-bottom Maxisorb (Nunc)plate wells were coated overnight with 0.2 μg of gag p24 HIV-1 (ImmunoDiagnostics, Inc.) diluted in 100 μl of coating buffer (15 mM Na₂CO₃, 35mM NaHCO₃ and 3 mM NaN₃, pH 9.6). The next day plates were blocked with200 μl of PBS containing 3% BSA for 2 hours. Serum samples were seriallydiluted in PBS supplemented with 3% BSA and in triplicates at 100μl/well on the gag-coated plates for 1 h at room temperature. Plateswere washed, and a 1:200 dilution of alkaline phosphatase-conjugatedgoat anti-mouse Igs (Cappel) was added to each well for 1 h at roomtemperature. After washing, plates were incubated with substrate (10 mgd-nitrophenyl phosphate disodium dissolved in 10 ml of 1 mM MgCl₂, 3 mMNaN₃, and 0.9 M diethanolamine, pH 9.8), and then read in an automatedELISA reader at 405 nm (model EL311. Bio-Tek Instruments).

Isolation of Lymphocytes

Peripheral blood mononuclear cells (PBMC), spleen and livers wereharvested as described (Lasaro et al., Microbes Infect 2005; 7:1541-50;Lin et al., Cancer Res 1996; 56:21-6). Tumor-infiltrating lymphocytes(TILs) were isolated from matrigel-tumors 3 or 10 days after challengeas described (22). TILs from the thyroid of E7-tg mice were harvestedupon treatment of thyroid tissue fragments with 2 mg/ml collagenase P(Roche) and 1 mg/ml DNase I (Invitrogen). After 1 hour, the thyroid washomogenized and filtrated through a 70-mm cell strainer. Cells werewashed with RPMI (Cellgro) media supplemented with 5% FBS, treated for 5min on ice with Ack lysing buffer (Invitrogen) to rupture red bloodcells, then suspended in 7 mL 40% percoll (Amersham Bioscience,Piscataway, N.J.), and applied on top of 3 mL 70% percoll. Aftercentrifugation at 2200 rpm for 20 min at room temperature, the cells atthe interface were harvested and resuspended in media.

Tetramer and Lymphocyte Markers Staining

Antigen-specific CD8⁺ T cells were detected by APC-labeled MHC class Itetramers carrying the AMQMLKETI peptide (SEQ ID NO:20) of gag or theRAHYNIVTF peptide of E7 (E7tet; SEQ ID NO: 19) (MHC Tetramer CoreFacility, Emory University Vaccine Center, Atlanta, Ga.). PBMC andsplenocytes isolated 10 days after immunization with AdC68 vectors weretreated as described for intracellular cytokine staining. Samples werestained for 30 minutes at room temperature with gag-tet andanti-CD8a-PerCP in combination with the following antibodies: CD25-PE,CD122-PE, CD127-PE, CD27-PE, BTLA-PE (eBioscience), PD1-PE, CD62L-FITC,CD69-FITC, CD103-FITC, CD43-FITC, CD44-FITC and CD54-FITC (all from BDBiosciences Pharmingen, unless indicated otherwise). For Bcl2 and CTLA-4staining, cells were washed, permeabilized for 30 min at 4° C. withCytofix/Cytoperm (BD Biosciences Pharmingen), and then stained withantibodies Bcl2-PE or CTLA-PE (BD Biosciences Pharmingen).

Lymphocytes were stained for 30 minutes at room temperature with E7-tetand anti-CD8a-PerCP together with the following antibodies: BTLA-PE(eBioscience), CD44-FITC, CD62L-PE, CD27-PE, CD127-PE, CD122-PE andPD1-PE (BD Biosciences Pharmingen). For Bcl2 and CTLA-4 staining, cellswere washed, permeabilized for 30 min at 4° C. with Cytofix/Cytoperm (BDBiosciences Pharmingen), and then stained with antibodies Bcl2-PE orCTLA-PE (BD Biosciences Pharmingen). Flow cytometry analyses wereperformed with at least 100,000 viable cells live-gated on FACSCaliburusing CellQuest software (BD Biosciences Pharmingen) and were analyzedusing FlowJo software version 7.1.2 (TriStar, Inc).

Example 2 Fusion Protein Constructs

The HPV-16/HSV-1 glycoprotein D (gD) chimeric gene, called gDE7E6E5, wascomposed of the complete open reading frame of gD, which hadincorporated into the ApaI site a fusion gene composed of HPV-16 E5, E6and E7 genes without respective start and stop codons (FIG. 1). TheHIV-1/gD chimeric gene, named gDgag, was composed of the complete openreading frame of gD which had incorporated into the NarI site acodon-optimized truncated form of gag HIV-1 clade B (FIG. 2).

Example 3 Effects of Fusion Protein Constructs on Host Immune System

Mice were immunized with 100 μg/mouse of DNA vaccines or 5×10¹⁰ virusparticles/mouse of E1-deleted adenovirus vectors. After 14 days (DNAvaccines) or 10 days (E1-deleted adenovirus vaccines) peripheral bloodmononuclear cells (PBMC) were stained for CD8 and IFN-γ by intracellularcytokine staining. Percentage represents number of CD8⁺/IFN-γ cells overtotal number of CD8⁺ cells.

Pre-challenge vaccination: ten days after immunization mice werechallenged with 5×10⁵ TC-1 cells (10 mice per group), in vitrotransformed syngeneic cells which express E7 and E6 and induce tumors inC57Bl/6 mice, and tumor growth was following per 60 days. Percentagerepresents number of tumor-free mice over total mice challenged at day60.

Post-challenge vaccination: Mice (10 per group) were vaccinated 5 daysafter challenged with 5×10⁵ TC-1 cells. Tumor growth was followed for 60days.

The results are shown in Table 3. Percentage represents number oftumor-free mice over total mice challenged at day 60. The fusion genegDE7E6E5 expressed by a DNA vaccine (pgDE7E6E5) or an E1-deletedchimpanzee-derived adenovirus vector (AdC68gDE7E6E5) induced highfrequencies of E7-specific CD8⁺ T cells and complete protection againsttumor cell challenge after a single dose. However, neither E7-specificCD8⁺ T cell responses nor protection to challenge were developed when gDwas not present, indicating that this fusion had dramatically improvedthe efficacy of the vaccines.

TABLE 3 Protection upon Protection upon E7-specific CD8⁺ pre-challengepost-challenge Immunization T cell response ^(b) vaccination ^(c)vaccination ^(d) pRE4 (gD only) 0.11% 0% 0% pE7E6E5 0.18% 0% 0%pgDE7E6E5 5.53% 100%  70%  AdC68gD 0.05% 0% 0% AdC68E7E6E5 0.05% 0% 0%AdC68gDE7E6E5 21.94% 100%  100% 

An E1-deleted chimpanzee-derived adenovirus carrying a codon-optimized,truncated form of gag HIV-1 clade B (AdC68gag) has been shown to inducea strong specific CD8⁺ T cell response (Fitzgerald et al. (2003) J.Immunol. 170:1416). However, gag-specific CD8⁺ T cell frequenciesinduced by the DNA vaccine (pgDgag) and the E1-deletedchimpanzee-derived adenovirus vector (AdC68gDgag) were higher when gagwas fused into gD (FIG. 3). The effect of gD fusion was more remarkablewhen lower amounts of adenovirus vector were used for vaccination (FIG.3B).

Although chimpanzee-derived adenovirus vectors circumvented the expectednegative effect of pre-existing immunity to common human serotypes ofadenovirus, such as serotype 5 (AdHu5), the efficacy of AdC68vaccination was decreased by approximately 50% when neutralizingantibodies to AdHu5 were present. Although not wishing to be bound bythis explanation, this reduction likely is caused by pre-existing T cellthat cross-react between adenovirus of the human serotype 5 and thechimpanzee adenoviruses. However, AdC68 vaccinations carrying antigensfused with HSV-1 gD were only weakly influenced by those antibodies(FIG. 4).

Example 4 Interaction of Constructs which Encode Elision Proteins withHVEM

Efficacy of the vaccine was related to the localization of the transgenein relationship the N-terminus of gD. The specific region localized inthe N-terminal portion of gD, which interacts with the herpes virusentry mediator (HVEM), was crucial in enhancing stimulation of specificCD8⁺ T cell mediated immune response. A mutated form of gD chimericprotein gDE7E6E5, called NBEFgDE7E6E5, was generated altering specificgD amino acids (M11A, N15A, L25, Q27A, L28A, T29A, D30A), which promotegD-HVEM interaction. The mutated form of gDE7E6E5 expressed by DNAvaccine was not able to induce an E7-specific CD8⁺ T cell response,although mutated and non-mutated gDE7E6E5 genes were transcribed at thesame level (FIG. 5). On the other hand, a single-amino acid modificationon HSV-1 gD (W294A), was able to enhance the efficacy of a gD/E7 fusionDNA vaccine (FIG. 6).

The interaction of HSV-1 gD with HVEM interferes with down-regulation ofimmune responses associated with HVEM-BTLA (B and T lymphocyteattenuator) pathway. Without intending to be bound by theory,maintenance of gD interference in HVEM-BTLA pathway is a important keyto enhance a specific T cell mediated immune response to one or moreheterologous antigens. To this end, it was demonstrated that a fusion ofan antigen, such as HIV-1 gag, into the C-terminal region of HSV-1 gDdid not decrease its affinity to bind to HVEM (FIG. 7). The ability ofHSV-1 gD to bind HVEM was not affected by fusion with HIV-1 gag. Infact, the affinity to HVEM of HIV-1 gag/HSV-1 gD chimeric protein wasslight higher than non-fused gD.

Confocal microscopy and FACS analyses of cells infected in vitro withAdC68gDgag provided additional evidence that gDgag bound HVEM (FIGS. 8and 9). Confocal microscopy indicated that gDgag was co-localized withHVEM on the membrane (FIG. 8A) and inside (FIG. 8B) cells infected withAdC68gDgag. Also, cell surface gD staining on AdC68gDgag-infected cellsdemonstrated that the amount of gD was greater on cells that expressedHVEM than on cells that did not express HVEM (FIG. 9). These resultsindicate that gDgag bound HVEM, that part of gDgag expressed by theinfected cell bound HVEM inside the cell, and that the complex ofgDgag-HVEM was transported from the inside the cell to the cell surface.In addition, confocal microscopy and gD staining indicate that gDgagencoded by AdC68gDgag-infected cell was able to bind to HVEM expressedon the surface of other non-infected cells (FIG. 10).

Human derived serotype 2 adeno-associated virus (AAV) vectors encodingthe gDgag chimeric protein induced higher CD8+ T cell response againstgag than AAV vector encoding only gag (FIG. 14). In addition,immunization with AAVgDgag enhanced CD8+ T cell response against AAVcapsid.

Example 5 CD8⁺ Tcell Response Against NP

The chimeric protein gDNP is composed of the complete open reading frameof HSV-1 gD, into which we incorporated into the ApaI site thenucleoprotein P (NP) gene from Influenza virus A/PR8 without respectivestart and stop codons (FIG. 15).

Mice were immunized with 100 μg of DNA vaccine vector pgDNP or pNP.Fourteen days after immunization peripheral blood mononuclear cells(PBMC's) were isolated and cell surface stained with the NP-tetramer anda labeled antibody to CD8. Naïve mice were used as negative control.Data are shown in FIG. 16 and represent percentages of NP-tetramer⁻ CD8⁺T cells over all detected CD8⁺ T cells. This experiment demonstratedthat the gDNP chimeric protein expressed by DNA vaccine vector inducedNP-specific CD8+ T cell response in mice after a single immunization.

Example 6 Molecular Modeling

To examine how insertion of a foreign sequence within the C terminus maymodify folding of gD we modeled the structure of unligated gD-gag (FIGS.18A, 18B) and of gD-gag bound to HVEM (FIGS. 18C, 18D). The C-terminusof the native unligated gD structure is anchored near the N-terminalregion and thus masks the HVEM binding site (Krummenacher et al., EMBOJ. 24, 4144-53, 2005; FIG. 19). However, computational modeling of thegD-gag structure predicts that the C-terminus would be shifted away fromthe N-terminal portion without altering the core structure or the Nterminus, which is required to form the HVEM binding site (FIGS. 18A,18B). A superposition of X-ray structure of gD upon the gD-gag modelindicates that insertion of gag into gD does not disrupt the integrityof the HVEM binding surface (FIGS. 18B, 18D).

Example 7 Cellular Localization of Chimeric gD Proteins

Binding of gD to HVEM presumably depends on secretion or cell surfaceexpression of gD. To determine the cellular localization of the chimericgD-gag protein, B78-H1/3E5 cells, which express human HVEM-EGFP on theirsurface, were infected with AdC68gag, AdC68gD or AdC68gD-gag, stainedwith a monoclonal antibody (MAb) to gD and analyzed by confocalmicroscopy (FIG. 19). Both gD and gD-gag co-localize with HVEM on thesurface of infected cells as well as within infected cells. Similarresults were obtained upon staining of cells with antibodies to gag.Absolute levels of gD-gag expressed on the surface of infected cellswere below those of native gD (FIG. 20). Analyses of mRNA levels showedthat all vectors transcribed the chimeric genes at equal amounts totheir corresponding non-chimeric versions. Thus the lower levels of cellsurface expression of the chimeric protein suggest inefficientsecretion, rapid re-internalization of gD-gag or accelerated proteolyticdegradation. The presence of HVEM on the cell surface increased theamount of gD-gag (FIG. 20) on the cell surface suggesting thatintracellular binding of HVEM to gD-gag stabilized gD-gag or facilitatedexport of the chimeric protein.

When AdC68gD or AdC68gD-gag infected HVEM negative (HVEM⁻) cells weremixed with uninfected cells expressing HVEM (HVEM), both gD and gD-gagco-localized with HVEM on the uninfected HVEM⁺ cells indicating thatsome of the protein was released and then bound to HVEM. This was morepronounced with gD-gag than with gD, and may reflect either increasedsecretion of the chimeric protein or its superior binding to HVEM (FIG.21). To ensure that the observed transfer of gD or gD-gag from HVEM⁻ toHVEM⁺ cells was not a result of cell fusion, cells were stained for anadditional marker, the coxsackie adenovirus receptor (CAR), expressedonly by the HVEM⁻ cell line. Uninfected HVEM⁺ cells which co-stained forgD failed to express CAR thus ruling out cell fusion.

Example 8 Expression of Antigens Within gD Augments Antigen-SpecificCD8⁺ T Cell Responses

To determine if expression of an antigen within gD affects stimulationof CD8⁺ T cells in general to epitopes presented on gD transducedantigen presenting cells we initially performed proliferation assays.Irradiated cells from draining lymph nodes of mice intramuscularly(i.m.) immunized with AdC68 carrying either gD or E7E6E5 were pulsedwith low amounts of the SIINFEKL peptide and served as antigenpresenting cells for carboxyfluorescein diacetate succinimidyl diester(CFSE)-labeled CD8⁺ T cells from OT-1 mice, which carry CD8⁺ T cellswith a transgenic receptor for SIINFEKL in the context of Kb. We hadshown previously that i.m. application of an AdC68 vector causes anaccumulation of vector transduced mature dendritic cells within draininglymph nodes²⁶. We thus expected that upon application of the AdC68gDvector some of the mature dendritic cells would express gD, which inturn may modulate the response of OT-1 derived CD8⁺ T cells to theircognate antigen. As shown in FIG. 22A, OT-1 CD8⁺ T cells proliferatedmore vigorously upon co-culture with lymph node cells from AdC68gDinjected mice than upon co-culture with cells from AdC68E7E6E5 injectedmice.

To evaluate if blockade of the HVEM inhibitory pathway enhances adaptiveimmune responses in vivo, we vaccinated mice with DNA and AdC68 vectorsexpressing the gD-antigen chimeric proteins and tested T and B cellresponses in comparison to those of mice injected with vectorsexpressing E7, E6 and E5 or gag without gD. Mice fail to mountdetectable E7-specific CD8⁺ T cell responses following vaccination withDNA or AdC68 vectors encoding either E7E6E5 (FIG. 22A) or E7 alone. Incontrast, the DNA vaccine and the AdC68 vector expressing E7 or thefusion polypeptide of E7, E6 and E5 within gD induce robust E7-specificCD8⁺ T cell responses. Similarly, the DNA vaccine expressing the gD-gagchimeric protein stimulate more potent gag-specific CD8⁺ T cellresponses compared to vectors expressing gag only (FIG. 22A). The AdC68vector expressing the gD-gag chimeric protein also elicits strongergag-specific CD8⁺ T cells than those expressing gag only, which wasespecially pronounced at low vector doses (FIGS. 22A, 22B). To determinethe longevity of vaccine-induced responses, E7-specific CD8⁺ T cellswere monitored for over a year following vaccination with theAdC68gD-E7E6E5 vectors. CD8⁺ T cell responses were maintained at stablefrequencies indicating that the enhancement of the initial primary Tcell response resulted in an increase of the memory T cell population(FIG. 22C).

Example 9 Expression of Antigens within gD Increases Antigen-SpecificAntibody Responses

It is well established that neutralizing antibodies are the primarycorrelate of vaccine-induced protection against most virus infections.LIGHT stimulation enhances both B cell proliferation and immunoglobulinproduction²⁷ and might balance inhibition exerted by ligation of BTLA toHVEM. To analyze whether antibody responses can be enhanced by thegD-antigen chimeric proteins, sera of mice immunized with the AdC68vectors expressing gag or gD-gag were analyzed for antibodies to gag(Table 4).

TABLE 4 Gag-specific antibody response after immunization with AdC68vectors expressing gD, gag, or gD-gag. Anti-gag antibody titer ± SD^(a)(p value)^(b) Immunization 10 days 7 weeks 20 weeks 49 weeks AdC68gag 26± 8 35 ± 8 152 ± 19 6 ± 4 AdC68gD 3 ± 4 (0.007) 9 ± 4 (0.0037) 28 ± 15(0.0004) 8 ± 2 (0.2573) AdC68gD-gag 287 ± 7 (2.6E−7) 740 ± 2 (6.8E−9)1257 ± 21 (4.8E−6) 294 ± 8 (3.3E−7) ^(a)Anti-gag Ig titers wereestablished as the reciprocal of the serum dilution that gave anadsorbance twice above that of the sera from naïve mice. SD-standarddeviation. ^(b)p-values were determined using one-tailed Student t testcomparing titers either from mice immunized with AdC68gD or AdC68gD-gagwith titers from mice immunized with AdC68gag.

The AdC68gag vector induces only marginal levels of gag-specificantibodies while AdC68gD-gag vector elicits a significantly higherresponse that remained detectable for at least 11 months. Antibodyresponses to gD, measured in parallel, were comparable in micevaccinated with the AdC68gD or the AdC68gD-gag vector.

Example 10 gD-HVEM Interaction is Needed to Augment Immune Responses

To determine if enhancement of the CD8⁺ T cell responses by expressionof an antigen within gD requires binding of gD to HVEM, we constructedDNA vaccines expressing the E7E6E5 sequence within two modified versionsof gD; In one construct, termed NBEFgD-E7E6E5, 7 amino acids at theN-terminus of gD. i.e. M11, N15, L25, Q27, L28, T29 and D30, werereplaced with alanine residues. Previous studies have shown that these 7amino acids on wild type gD are crucial to gD-HVEM interaction¹³. In asecond construct, termed SgD-E7E6E5, the tryptophan (W) in position 294of gD was changed to alanine (A). This modification, which is thought todestabilize the native conformation of the gD C-terminus, was shown toincrease binding of gD306 to HVEM¹⁵. Mice immunized with one dose of aDNA vaccine expressing NBEFgD-E7E6E5 failed to mount detectablefrequencies of E7-specific CD8⁺ T cells (FIG. 24A). In contrast,frequencies of E7-specific IFN-γ CD8⁺ T cells induced by pSgD-E7E6E5were higher compared to those induced by pgD-E7E6E5 (FIG. 24A), althoughthis difference did not reach statistical significance. To confirm thisobservation we also modified the pgD-E7 DNA vaccine²⁸, which carriesonly E7 inserted into gD, by changing the tryptophan in position 294 toalanine. This new vector, termed pSgD-E7, stimulated significantlyhigher frequencies of E7-specific CD8⁺ T cells compared to a plasmidvector expressing E7 within the wild-type form of gD (FIG. 24B). Takentogether, these results demonstrate that binding to HVEM is essentialfor the immunopotentiating effect of gD and that gD mutations thatincrease its binding to HVEM can under some conditions further augmentCD8⁺ T cell responses.

Example 11 Functionality of CD8⁺ T cells Induced by gD Chimeric Proteins

Modulation of regulatory pathways may affect the functionality of theresultant T cell responses. Phenotypes of vaccine-induced CD8⁺ T cellswere analyzed from mice immunized with AdC68 vectors expressing gD-gagor gag (FIG. 25). We measured expression of the following phenotypicmarkers, i.e., CD25, CD122, CD127, CD27, CD62L, CD69, CD103, CD43, CD44,CD54, Bcl2, BTLA, CTLA-4 and PD-1 on gag-specific CD8⁺ T cells (FIG.25). Most of the markers tested (CD122, CD127, CD27, CD62L, CD69, CD103,CD43, CD44, CD54, Bcl2, BTLA, CTLA-4 and PD-1) are modulated onantigen-specific CD8⁺ T cells as compared to naïve CD8⁺ T cells and formost markers expression levels on CD8⁺ T cells induced by gag or gD-gagare identical. CD27 is increased on a subpopulation of gD-gag inducedgag-specific CD8⁺ T cells, while CTLA expression is marginally lowerwhen compared to CD8⁺ T cells induced by gag alone. Overall, althoughAdC68gD-gag elicits higher frequencies of gag-specific CD8⁺ T cells thanAdC68gag, the phenotypic profiles of the resultant effector cells arevery similar. IFN-γ⁺ CD8⁺ T cells induced by gD-gag or gag respond tothe same epitope when tested against a panel of gag peptides, indicatingthat expression of gag within gD does not increase the breadth of theCD8+ response.

T cell functionally was further accessed by testing if mice vaccinatedwith either DNA (FIG. 26A) or AdC68 vectors (FIG. 26B) expressing E7E6E5with or without gD were protected against challenge with TC-1 cells,which are lung epithelial cells derived from C57Bl/6 mice that aretransformed with v-Ha-ras and the E6 and E7 oncoproteins of HPV-1626.Animals immunized with pgD-E7E6E5 or AdC68gD-E7E6E5 were completelyprotected against TC-1 tumor progression, and protection was only seenupon vaccination with constructs carrying gD fusion proteins. Micevaccinated with DNA vaccines expressing E7 or E7E6E5 within mutant formsof gD were also tested for protection against TC-1 tumor formation (FIG.26C). Protection correlated with CD8⁺ T cell responses: mice immunizedwith a vector expressing E7E6E5 within the form of gD that can not bindto HVEM were not protected while mice immunized with the same antigenexpressed within the gDW294A variant were fully protected. Additionally,animals immunized once with a DNA vaccine expressing only E7 within gDdeveloped tumors while those that expressed E7 within the gDW294Avariant were completely protected.

Discussion of Examples 2-11

Incorporation of the antigens into the extracellular C-terminal domainof gD markedly increases vaccine-induced CD8⁺ T and B cell responses. Asdemonstrated in the specific examples, above, this is especiallypronounced for CD8⁺ T cell responses to E7, which expresses a T cellepitope with low to moderate affinity to H-2 Kb (He et al., virol. 270,146-61, 2000). A single dose of DNA vaccines or AdC68 vectors expressingthe E7E6E5 polypeptide fails to elicit detectable CD8⁺ T cell responsesor protective immunity to challenge with an E7-expressing tumor cellline, while the same antigen expressed within gD by either vectorresults in high and sustained frequencies of E7-specific CD8⁺ T cellsand complete protection against tumor cell challenge. Gag of HIV-1carries a high affinity epitope for mice of the H-2^(d) haplotype andDNA vaccines or AdC68 vectors (Fitzgerald et al., J. Immunol. 170,1416-22, 2003) expressing gag induce readily detectable CD8⁺ T cellresponses. Responses to gag are also increased upon expression of gagwithin gD.

The immunopotentiating effect of gD on the response to gag is moreimpressive when the gD-gag chimeric protein is delivered by a DNAvaccine rather than by the highly immunogenic AdC68 vector. Upon dosereduction of the AdC68 vector, results clearly demonstrate that aninduction of gag-specific CD8⁺ T cells can be achieved with anapproximately 100 fold lower vaccine dose than needed for the AdC68vector expressing gag only. This is important because Ad vectors of thecommon human serotype 5 (AdHu5) when tested in clinical trials asvaccine carriers for antigens of HIV-1 encountered dose limitingtoxicity (Kresge, IAVI rep. 9, 18-20, 2005). Reactogenicity at highdoses in humans is also anticipated with the chimpanzee-origin Advectors such as AdC68, which was developed to circumvent the effect ofneutralizing antibodies to common human serotypes of adenovirus. Thus,further improvement of the immunogenicity of Ad vector vaccines thatallows for a substantial dose reduction while maintaining efficacy couldlower vaccine-related side effects, reduce the overall cost of thevaccine and facilitate production for mass vaccination. Increasedefficacy may also lessen the need for complex prime boost regimens thatare currently being tested in clinical HIV-1 vaccine trials but that maybe unmanageable and too costly for developing countries.

Insertion of antigens into gD does not affect the functionality ofantigen-specific CD8⁺ T cells; T cells induced by the gD chimericproteins protect against tumor challenge in the E7 model, arephenotypically similar to those induced in absence of gD, andefficiently differentiate into memory cells as shown with gag-expressingAdC68 vaccines, confirming a previous study with BTLA-deficient T cells(Krieg et al., Nat. Immunol. 8, 162-71, 2007).

Antibody responses are also augmented by expressing the antigen withingD. LIGHT participates in B cell expansion as a co-stimulus of CD40 andinduces antibody production (Duhen et al. Eur. J. Immunol. 34, 3534-41,2004). The control of LIGHT-induced B cell activation appears to beprovided by down-regulation of HVEM following its engagement by LIGHT(Duhen et al., 2004). BTLA is constitutively expressed on B cells andBTLA deficient mice mount higher antibody responses compared towild-type mice although this effect was shown previously to be modest(Hurchla et al., J. Immunol. 174, 3377-85, 2005). In contrast, in ourvaccine model, antibody responses were markedly enhanced upon expressinggag within gD. While not wishing to be bound by this explanation, thismay reflect that binding of gD to HVEM augments CD4⁺ T cell responses,which in turn promote activation of B cells. Alternative pathways suchas an enhancement of the co-stimulatory HVEM-LIGHT pathway may also havecontributed.

Binding of gD to HVEM is essential for augmentation of CD8⁺ T cellresponses to the fusion partner as vaccines expressing antigen within amodified gD in which the HVEM binding site had been obliterated fail toinduce enhanced CD8⁺ T cell responses. In the native unligated structureform of gD, the C-terminus largely obstructs movement of the N-terminusinto its HVEM-binding conformation (Krummenacher et al., EMBO J. 24,4144-53, 2005). According to our molecular model, insertion of foreignsequences of certain lengths such as gag or the E7E6E5 polypeptide mayeffect a structural change of gD which improves its binding to HVEM.This may not be achieved by short sequences such as E7 alone, for whichthe immunogenicity and efficacy of the gD chimeric vaccines can beimproved further through a single amino acid exchange in position 294 ofgD which had been described previously to improve binding of gD to HVEM(Krummenacher et al., 2005).

Manipulation or disruption of negative regulatory pathways has beenshown previously to augment T cell responses. Recent reports showed thatantibody-mediated interruption of the PD1-PD-L1 pathway reverses T cellexhaustion caused by chronic infections and allows for increased T cellproliferation (Barber et al. Nature 439, 682-87, 2006: Day et al. Nature443, 350-54, 2006). Manipulation of this pathway may not readily affectprimary T cell responses as PD1 requires induction by activation and isnot expressed on naïve T cells (Barber a al., 2006). In contrast, lowlevels of BTLA are expressed on naïve T cells: the levels rapidlyincrease upon T cell activation and then decline (Hurchla et al., 2005;Han et al., J. Immunol. 172, 5931-39, 2004).

Medicinal targeting of immunoregulatory pathways can result inimmunopathology such as the devastating cytokine storms observed uponapplication of anti-CD28 antibodies to human volunteers (Suntharalingamet al., NEJM 355, 1018-28, 2006) or auto-immunity commonly seen ingenetically modified mice (Watanabe et al., Nat. Immunol. 4, 670-79,2003). The use of a gD-antigen chimeric protein to enhance theimmunogenicity of vaccines has the advantage that it does not involvesystemic interruption of an inhibitory pathway but rather exerts itseffects locally to the site of antigen presentation. We confirmed thespatially limited effect of gD experimentally by injecting two Advectors expressing either gD or E7E6E5 into distant anatomical sites(left leg versus right leg) and failed to observe enhancement ofantigen-specific CD8⁺ T cell responses. It has been suggested previouslythat targeting of BTLA through inhibitory antibody or small moleculesmay enhance vaccine immunogenicity. Results shown here suggest that suchnovel adjuvants may indeed be useful; nevertheless, their effect, unlikethat of gD chimeric antigens, would be systemic and thus carry a higherlikelihood of unwanted side effects.

Example 12 In Vitro Characterization of the Vaccine Vectors

We compared levels of transgene in cells infected in vitro with AdC68vectors. Total RNA isolated from CHO/CAR cells infected with 109 virusparticles (vps) of AdC68gD, AdC68E7E6E5 and AdC68gD-E7E6E5 vectors wasreversed transcribed and gD, E7, E6 and E5 specific mRNA were quantifiedby real-time PCR (FIG. 27A). To minimize inherent differences in RNAisolation procedures, mRNA of a housekeeping gene (GAPDH) was used tonormalize the amount of cDNA in each sample. The amount of gD-specificmRNA transcripts in cells infected with AdC68gD-E7E6E5 was notstatistically different from that in cells infected with AdC68gD. ThemRNA levels of E7, E6 and E5 were also similar in samples infected withAdC68gD-E7E6E5 and AdC68E7E6E5. Protein expression in cells infectedwith AdC68 vectors was evaluated by immunofluorescence with a monoclonalantibody (MAb) against gD (FIG. 27B). CHO/CAR cells infected withAdC68gD expressed gD mainly on their surface although some of theprotein could be detected within the cells. Cells infected withAdC68E76E5 did not stain with the gD-specific MAb. In cells infectedwith AdC68gD-E7E6E5, gD was mainly detected within the cells; levels ofgD on the cell surface were markedly reduced when compared to those oncells infected with AdC68gD. The lower levels of cell surface expressionof the chimeric protein might be due to inefficient secretion, rapidre-internalization of gD-E7E6E5 or accelerated proteolytic degradation.

Example 13 E7-specific CD8⁺ T Cell Responses Induced by AdC68 Vectors

The induction of immune responses by the gD-E7E6E5 expressed by AdC68and DNA vectors were described above. Here, we confirmed our studies bytesting splenocytes and PBMCs from mice immunized with either AdC68 orDNA by ICS for frequencies of CD8⁺ T cells producing IFN-γ in responseto a peptide expressing the immunodominant epitope of E7 (FIGS. 28A and28B, respectively). We confirm that immunization with AdC68gD-E7E6E5induced a potent E7-specific CD8⁺ T cell response detectable fromspleens and blood while no specific CD8⁺ T cell response was found uponimmunization with AdC68gD or AdC68E7E6E5. Also, DNA vaccines carryingeither the E7E6E5 fusion protein alone or gD failed to induce CD8⁺ Tcells to E7, while such cells were elicited by the DNA vaccineexpressing E7E6E5 within gD.

To investigate whether the CD8⁺ T cell response induced byAdC68gD-E7E6E5 could be enhanced further, AdC68gD-E7E6E5 was tested in aprime and boost regimen with the DNA vaccine expressing the sametransgene product (FIG. 28C). Groups of mice were primed with the DNAvaccine and then boosted with the AdC68 vector 90 days later. In otheranimals the order of the vaccines was reversed. T cell frequencies weretested from blood 14, 30 and 90 clays after priming and then on days 7,10 and 14 after the boost. After priming, the AdC68 vector inducedhigher frequencies of E7-specific CD8⁺ T cells compared to the DNAvaccine. Upon booster immunization the AdC68 prime DNA boost regimenperformed poorly, and although CD8⁺ T cell frequencies increased theyremained below the peak frequencies seen upon AdC68 priming. Incontrast, in the group primed with the DNA vaccine, a boost with theAdC68 vector induced a pronounced increase in E7-specific CD8⁺ T cellfrequencies.

Once we defined the most efficient prime boost regimen, we testedwhether the DNA vaccine or the AdC68E7E6E5 vector could prime or boost aCD8⁺ T cell response to E7. Mice primed with the DNA vaccines carryingE7E6E5 with or without gD were boosted 90 days later with eitherAdC68E7E6E5 or AdC68gD-E7E6E5, respectively. See Table 5, below.

TABLE 5 AdC68 vectors boost after prime with DNA vaccines. Percentage ofIFN-γ CD8+ cells over total CD8+ cells ^(b) Boost ^(c) Prime ^(a) NoBoost AdC68E7E6E5 AdC68gD-E7E6E5 No prime 0.1 0.1 21.9 pE7E6E5 0.1 0.121.2 pgD-E7E6E5 0.3 2.5 47.4 ^(a) Mice were primed i.m. immunized with100 μg of DNA vaccine. ^(b) Frequencies of E7-specific CD8+ T cells overall CD8+ cells were determined 10 days after boost. ^(c) Mice wereboosted with 5 × 10¹⁰ vps of AdC68 vectors 60 days after prime.

Ten days after immunization with AdC68gD-E7E6E5, E7-specific CD8⁺ T-cellfrequencies were similar in mice primed with pE7E6E5 and imprintedcontrol mice, indicating that the DNA vaccine expressing theoncoproteins of HPV-16 was non-immunogenic. Again, mice primed withpgDE7E6E5 and boosted with AdC68gD-E7E6E5 developed high frequencies ofE7-specific IFN-γ-producing CD8⁺ T cells. A low but significant increasein E7-specific CD8⁺ T cells was observed in mice primed with the DNAvaccine expressing gD-E7E6E5 and then boosted with AdC68E7E6E5,indicating that within this more immunogenic vaccine vehicle, theoncoproteins could trigger expansion of a memory response.

To test whether the effect of gD required that the antigen was expressedwithin gD or if concomitant presence of gD and the antigen sufficed, weimmunized mice with a mixture of equal doses of AdC68gD and AdC68E7E6E5.This mixture failed to elicit a detectable E7-specific CD8⁺ T cellresponse, suggesting that the immunopotentiating effect of gD requiresthat the antigen is expressed within gD or that the same cells thatexpress gD have to express the antigen.

Example 14 Dose Response Curve of the CD8⁺ T Cell Response toAdC68gD-E7E6E5

In humans, Ad vectors cause dose limiting toxicity, as has beenestablished for E1-deleted AdHu5 vector tested as vaccine carriers forantigens of HIV-1 in clinical trials (Kresge, K. J et al., 2005).Although the AdC68 vector has not yet undergone clinical testing, weanticipate that this vector would also cause significant toxicity ifused at high doses. We therefore tested the E7-specific CD8⁺ T cellresponse elicited by varied doses of AdC68gD-E7E6E5 (FIG. 29A).

Mice were immunized i.m. with 1×10⁸ to 5×10¹⁰ vps of AdC68gD-E7E6E5.Frequencies of E7-specific CD8⁺ T cells were measured 10 days later fromblood and spleens. The specific CD8⁺ T response declined with decreasingdoses of adenovirus vector. A response could still be detected uponimmunization with 5×10⁸ vps of the vaccine but then became undetectableat 1×10⁸ vps. Comparable results were obtained with other immunogens, asdescribed above.

Example 15 AdC68gD-E7 E6E5 Protects Against Challenge With anE7-Expressing Tumor Cell Line

To formally demonstrate efficacy of AdC68gD-E7E6E5 we conductedchallenge experiments with the TC-1 cell line. To determine theeffectiveness of the AdC68 vector-induced immune response in causingregression of already established tumors, groups of C57Bl/6 mice werefirst injected with TC-1 cells then vaccinated 5 days later withAdC68gD, AdC68E7E6E5, or AdC68gD-E7E6E5 (FIG. 30A). Mice immunized withAdC6SgD-E7E6E5 rejected the tumors and remained tumor-free for at least60 days after challenge. In contrast, mice immunized with AdC68gD andAdC68E7E6E5 showed progressive growth of the TC-1 tumors.

Induction of long-term memory by a therapeutic vaccine is advantageous,since longevity of T cells may prevent resurgence of virus-infectedcells that escape the initial wave of the immune response. Recently, weshowed that E7-specific CD8⁺ T response in mice immunized with a singledose of AdC68gD-E7E6E5 was detected over a year after immunization. Wechallenged mice that had been vaccinated 1 year earlier withAdC68gD-E7E6E5, AdC68gD or AdC68E7E6E5 with TC-1 cells (FIG. 30B). Miceimmunized with AdC68gD-E7E6E5 were protected against TC-1 challengegiven one year later, while mice injected with AdC68gD or AdC68E7E6E5developed tumors after challenge.

To determine if tumor cell challenge boosted the vaccine-inducedE7-specific CD8⁺ T cell response, we investigated E7-specific CD8⁺ Tcell response after challenge (FIG. 31). Frequencies of E7-specific CD8⁺T cells as detected by E7/D^(b) tetramers increased slightly in micethat were challenged compared to those that had only been vaccinated(FIGS. 31A and 31B).

Example 16 Phenotypic Profile of E7-Specific CD8⁺ T Cells

We analyzed CD8⁺ T cells from blood, spleens and rumors of vaccinatedmice. Mice were immunized with AdC68gD, AdC68E7E6E5 or AdC68gD-E7E6E5.They were challenged 10 days later with TC-1 cells in matrigel.Lymphocytes were isolated 3 days later from spleens, blood and tumors. Apronounced cellular infiltrate was seen in tumors from AdC68gD-E7E6E5vaccinated animals while comparatively few cells could be isolated fromtumors of the other groups. We therefore isolated cells from additionalmice on day 10 after challenge. At this time point there was apronounced infiltrate in the tumors of AdC68gD and AdC68E7E6E5vaccinated mice. In mice vaccinated with AdC68gD-E7E6E5, tumors hadresolved and only a few cells could be recovered. We therefore comparedcells isolated from day 3 tumors from AdC68gD-E7E6E5 vaccinated micewith those isolated on day 10 from tumors of AdC6SgD and AdC68E7E6E5vaccinated mice. In addition we analyzed PBMCs and splenocytes harvestedon the corresponding days.

Tumors from mice vaccinated with AdC68gD-E7E6E5 had frequencies ofE7-specific CD8⁺ T cells that exceeded those in spleens or blood of thesame mice, indicating a rapid recruitment or retention of E7-specificCD8⁺ T cells within the tumors. E7-specific CD8⁺ T cells from spleens,blood and tumors were analyzed phenotypically for CD44, CD62L, CD27,Bcl2, BTLA, CTLA-4 and PD-1 in comparison to tetramer negative (tet−) Tcells from naïve mice or mice immunized with AdC68gD or AdC68E7E6E5. Thephenotypic profiles of tet− T cells from either of these groups wereidentical. E7-specific CD8⁺ T cells from spleens and blood up-regulatedCD44 and down-regulated CD62L. CD27, Bcl2 and BTLA. There was no changeof CTLA-4 or PD1 expression compared to tet-CD8⁺ T cells. CD8⁺ T cellsisolated from tumors showed a distinct phenotype. Tet CD8⁺ cellsup-regulated CD44 and down-regulated CD62L, CD27 and BTLA. Unlike cellsfrom blood and spleens, they failed to down-regulate Bcl2 and stronglyupregulated CTLA-4 and PD1, two molecules involved in negativeimmunoregulation.

Example 17 AdC68gD-E7E6E5 Induces an E7-Specific CD8⁺ T Cell Response inE7-Transgenic Mice

Women with HPV-16-associated cancers are expected to respond poorly toE7 as a progressing tumor would impair the adaptive immune responsedirected against its antigen. To determine if AdC68gD-E7E6E5 induces anE7-specific CD8⁺ T cell response in mice that are tolerant to E7 wetested transgenic (tg) mice that constitutively express E7 under atissue-specific promoter in the thyroid. These mice develop with agelarge goiters and thyroid carcinomas. E7-tg mice as well as age-matchedcontrol mice were vaccinated at one year of age with 5×10¹⁰ vps ofAdC6SgD-E7E6E or as a control with AdC68gD or AdC68E7E6E5. Ten dayslater lymphocytes were isolated from blood and spleens.

E7-tg mice had markedly enlarged thyroids and lymphocytes were alsoisolated from their thyroids. The thyroids of wild-type mice werecomparatively small and we were unsuccessful to obtain lymphocytes fromthem. T cells from individual animals were tested for frequencies ofE7-specific CD8⁺ T cells. Mice failed to develop E7-specific CD8⁺ Tcells upon vaccination with AdC68gD or AdC68E7E6E5 (FIG. 33A). BothE7-tg and wild-type mice developed vigorous responses in blood andspleens to E7 upon vaccination with AdC68gD-E7E6E5. Responses wereslightly higher in samples from wild-type mice compared to those fromE7-tg mice. This difference was statistically not significant by ICSalthough it was significant upon analysis by tetramer staining.E7-specific CD8⁺ T cells could also be detected at high frequencies inthe thyroid of AdC6SgD-E7E6E5-vaccinated E7-tg mice. E7-specific CD8⁺ Tcells isolated from blood and spleen of wild-type and E7-tg mice as wellas from thyroids of E7-tg mice were analyzed for expression ofphenotypic markers.

CD8⁺ T cells from naïve E7-tg mice isolated from spleens, blood orthyroids were analyzed for comparison. In spleen and blood expression ofCD62, CD127. BcL2, BTLA, and CTLA-4 on E7-specific CD8⁺ T cells fromE6/E7-tg and wild-type mice were indistinguishable. CD27. CD44 and PD-1were slightly higher on E7-specific CD8⁺ T cells isolated from blood andto a lesser degree from spleens of E7-tg mice. E7-specific CD8⁺ T cellsisolated from the thyroids expressed markedly higher levels of CD44,CD27 and PD-1 in comparison to CD8⁺ T cells isolated from the thyroidsof naïve E7-tg mice. The other markers were similar to those seen onE7-specific T cells from blood and spleens. None of the CD8⁺ T cellsisolated from the thyroids of naïve E7-tg mice stained with the tetramerto E7, phenotypes of the vaccine-induced E7-specific CD8⁺ T cells couldthus not be compared to phenotypes of T cells induced by the transgenicE7 protein.

Discussion of Examples 12-17

A preventative vaccine for HPV-16 based on L1 virus like particles,which induces neutralizing antibodies, has recently been licensed by FDAand has been recommended for use in teenage girls and young women. Oncean infection has occurred, however, neutralizing antibodies do notaffect viral clearance nor do they inhibit development of malignancies.Consequently, women with humoral immunodeficiency do not have anincreased susceptibility to cervical cancer, while women withcell-mediated immunodeficiency, such as HIV-1-infected patients, renaltransplant patients or patients with genetic T cell deficiencies, haveincreased incidence rates (Moscicki et al., J Infect Dis 2004;190:37-45; Matas et al. Lancet 1975; 1:883-6; Frisch et al., J NatlCancer Inst 2000; 92:1500-10; Lowy et al, J Natl Cancer Inst 2003;95:1648-50. This together with extensive studies in animal models andclinical trials (reviewed in Galloway, Lancet Infect Dis 2003; 3:469-75)implicates a crucial role for T cells in eliminating cells persistentlyinfected with oncogenic types of HPVs.

The vaccine described here expressed three of the oncoproteins ofHPV-16, i.e., E7. E6 and E5 to broaden responses in human outbredpopulations. Mice of the H-2^(b) haplotype respond to a low affinityepitope of E7, but according to our results fail to develop CD8⁺ T cellsto E6. Our CD8⁺ T cell analyses thus focused on responses to E7. Toenhance immune responses we incorporated the oncoproteins into gD ofHSV-1 which binds to the herpes virus entry mediator (HVEM) (Montgomeryet al. Cell 1996:87:427-36; Whitbeck et al., J Virol 1997; 71:6083-93).HVEM, which is expressed on dendritic cells, is a member of thetumor-necrosis factor receptor (TNFR) family and interacts with LIGHT(Marsters et al., J Biol Chem 1997; 272:14029-32; Granger & Rickert,Cytokine Growth Factor Rev 2003; 14:289-96) and lymphotoxin-α (LT-α)(Mauri et al. Immunity 1998; 8:21-30; Sarrias et al., Mol Immunol 2000;37:665-73). Also, HVEM binds to B and T lymphocyte attenuator (BTLA), arecently described member of the B7-family (Gonzalez et al., Proc NatlAcad Sci USA 2005; 102:1116-21; Sedy et al., Nat Immunol 2005; 6:90-8).The HVEM-BTLA interaction inhibits T cell activation in vitro thusdefining these molecules as part of an inhibitory pathway (Sedy et al.,2005). Expression of BTLA is upregulated on tumor-infiltrating T cellsas was shown in cancer patients (Wang et al. Tissue Antigens 2007;69:62-72). HSV-1 gD competes with BTLA for binding to HVEM (Compaan etal., J Biol Chem 2005; 280:39553-61) and would thus be expected toenhance activation of naïve T cells by blockade of this negativeimmunoregulatory pathway. Recently, we showed that viral antigensexpressed within gD induced CD8⁺ T and B cell responses to the antigensthat are far more potent than those elicited by the same antigenexpressed without gD.

Our data confirm a very strong increase of CD8⁺ T cell responses to E7expressed within HSV-1 gD. Responses to E7 expressed by the DNA vaccineor the AdC68 vector without gD were below the level of detection, whileE7 expressed within gD induced frequencies of E7-specific CD8+ T cellsof 1-3% upon DNA vaccination and 10-24% upon Ad vector immunization. Asexpected, responses were markedly higher upon vaccination with the AdC68vector than the DNA vaccine. Most of our studies therefore focused onthe AdC68 vector vaccine. Mice vaccinated with AdC68gD-E7E6E5 werecompletely-protected against challenge with the E7 and E6 expressingTC-1 tumor cells given shortly after vaccination or one year later. Moreimportantly, mice with pre-existing TC-1 tumors rejected the tumors uponvaccination and then remained disease-free. Vaccine-induced E7-specificCD8⁺ T cells rapidly enriched within the TC-1 tumors. PhenotypicallyE7-specific CD8⁺ T cells isolated from TC-1 tumors upregulated CD27,CTLA-4 and PD-1 and down-regulated Bcl2, which may have been aconsequence of the engagement of their receptors by the tumor cells.Expression of BTLA, which was previously reported to become upregulatedon TILs cells in humans, was not increased on E7-specific CD8⁺ T cellsisolated from TC-1 tumors.

Numerous studies have shown efficacy of HPV-16 E7 vaccines against TC-1tumors. Transplantable tumors grow very rapidly in mice and vaccines arethus applied before or shortly after challenge. At this early stage, Tcells to the tumor antigens are probably not yet compromised. Wetherefore tested AdC68gD-E7E6E5 in E7-tg mice, which express theoncoprotein under a tissue specific promoter within their thyroid.Listeria based E7 vaccines have been tested in E6/E7-tg mice in whichthe transgenes were similarly expressed with a bovine thyroglobulinpromoter (Souders et al., Cancer Immun 2007:7:2). The Listeria vaccinewas shown to induce lower frequencies of E7-specific CD8⁺ T cellscompared to wild-type mice and the average avidity of CD8⁺ T cells thatwere induced was ten fold lower than those isolated from wild-type mice.Nevertheless the Listeria vaccine could eradicate 7 day established (5mm) transplanted tumors in some E6/E7 transgenic mice, albeit at lowerfrequency than in wild-type mice. We tested AdC68gD-E7E6E5 inone-year-old E7-tg with thyroid hyperplasia. AdC68gD-E7E6E5 induced anE7-specific CD8⁺ T cell response in the E7-tg mice that was onlyslightly below that induced in age-matched wild-type mice. Thevaccine-induced E7-specific CD8⁺ T cells infiltrated the thyroid.Phenotypically, the thyroid-infiltrating vaccine-induced E7-specificCD8⁺ T cells in E7-tg mice showed only minor difference to thoseisolated from TC-1 tumors and these differences, i.e., an overalldecrease in CD44, CD27, Bcl2, and CTLA4 may be a reflection ofdifference in sampling time rather than T cell functionality.Accordingly E7-specific CD8⁺ T cells isolated from blood or spleens ofvaccinated wild-type or E7-tg mice showed virtually identicalphenotypes.

Our studies show that E7 of HPV-16 expressed as a fusion proteintogether with E6 and E5 within gD induces a robust CD8⁺ T cell responseeven in animals with developing E7-associated malignancies. Withoutfurther studies directly comparing the effect of similar numbers ofE7-specific CD8⁺ T cells induced by a vaccine expressing E7 without gDto those induced by a vaccine expressing E7 within gD we can onlyspeculate that the high efficacy of the latter is linked to blockade ofan immunoinhibitory pathway. The Listeria vector used previously inducesfrequencies of E7-specific CD8⁺ T cells that in wild-type mice arecomparable in magnitude to those induced by AdC68gD-E7E6E5. In E6/E7-tgmice the Listeria vector induced markedly lower frequencies of T cells(Souders et al., 2007), while the response to AdC68gD-E7E6E5 is onlyslightly reduced. Although it is tempting to speculate that this iscaused by an immunopotentiating effect of gD, other differences in thevaccine delivery vehicles such as their interactions with antigenpresenting cells, in addition to differences between the E6/E7 and E7transgenic mice such as transgene copy number and E7 expression may alsohave contributed to these results.

Blockade of immunoinhibitory pathways that are upregulated in cancerpatients such as regulatory T cells or PD-1 expression on T cells hasbeen shown pre-clinically to increase T cell responses to tumorantigens. In these systems regulatory T cells were depleted or rendereddysfunctional by antibodies and using a similar approach, the PD-1pathway was blocked by antibodies to PD-1 or its ligand. Theseinterventions, which have shown promise in animal models, exert a globaleffect on the immune system, which poses the risk of augmentingauto-immune reactivity. In contrast, blockade of the BTLA-HVEM pathwayonly exerts a local effect on T cells that are being activated to theantigen expressed within gD and should thus not subject patients tounwanted immune responses.

Example 18 Immunization of Rhesus Macaques

Two groups of rhesus macaques (4 per group) are enrolled into the study.Animals are screened for antibodies to AdC68. Only seronegative animalsare used. Sera and peripheral PBMCs are harvested 4 and 2 weeks beforevaccination and preserved to serve as controls.

Animals are vaccinated once with 500 μg of pgag (group 1) or pgD-gag(group 2). They are boosted 2 months later with 5×10¹⁰ vps of purifiedand quality controlled AdC68gag (group 1) or AdC68gD-gag (group 2)vector given i.m. in saline. Animals are bled 2, 4, and 8 weeks afterpriming and 2, 6 and 12 weeks after booster vaccination. PBMCs aretested for T cell responses to a pool of gag peptides by ELISpot forIFN-γ and IL-2 and by ICS for CD3, CD8, CD4, IFN-γ and IL-2 as described(Reyes-Sandoval, et al. J. Virol. 78:7392-7399).

Sera are tested for antibodies to gag (ELISA) and neutralizingantibodies to the vaccine carrier. The experiments are controlled bysamples collected prior to vaccination. Animals are euthanized ˜4 monthsafter the boost, and lymphocytes are isolated from various compartments(spleen, blood, lymph nodes, liver, intestine) and tested by ELISpot forT cell responses to gag including analyses for IFN-γ, IL-2. TNF-α andMIP-1β. In addition they are analyzed by ICS for secretion of IFN-γ andexpression of T cell markers (CD3, CD4, CD8, included into each panel),activation (CD69. CD25. CD95, CD71) and proliferation (Ki67) markers,markers that identify T cell subsets (naive, central memory T cells,effector memory T cells and effector T cells) (CD28, CD95. CD45RA,CD62L, CCR7, CD27, CD127); chemokine and homing receptors (CCR5, CCR9,CXCR4, C11a-c, a4b7, CD103, CD49d); and markers indicative for lyticpotential (CD107, perforin, granzyme B).

Immunophenotypical studies are performed by multicolor (7-8 colors) flowcytometry on mononuclear cells. The analyses are restricted tocompartments that that allow for isolation of sufficient numbers oflymphocytes. The experiment is controlled using cryopreservedsplenocytes from sham-vaccinated rhesus.

It is to be understood that the embodiments of the present inventionwhich have been described are merely illustrative of some of theapplications of the principles of the present invention. Numerousmodifications may be made by those skilled in the art based upon theteachings presented herein without departing from the true spirit andscope of the invention.

The invention claimed is:
 1. A nucleic acid molecule which encodes afusion protein, wherein the fusion protein comprises: (1) a firstpolypeptide segment comprising at least amino acids 1-240 of a matureHerpes simplex virus (HSV) glycoprotein D, wherein the first polypeptidesegment does not comprise a full length mature glycoprotein D; (2) asecond polypeptide segment comprising at least one antigen, wherein theat least one antigen is not an HSV glycoprotein D antigen, wherein the Nterminus of the second polypeptide segment is linked to the C terminusof the first polypeptide segment; and (3) a third polypeptide segmentcomprising a C terminal portion of the HSV glycoprotein D, wherein the Nterminus of the third polypeptide segment is linked to the C terminus ofthe second polypeptide segment, wherein the at least one antigen isselected from the group consisting of: an influenza virus antigen; anucleoprotein P influenza virus antigen; a Plasmodium antigen; aPlasmodium antigen selected from the group consisting ofthrombospondin-related anonymous protein (TRAP), ring-infectederythrocyte surface antigen (RESA), merozoite surface protein 1 (MSP1),merozoite surface protein 2 (MSP2), merozoite surface protein 3 (MSP3),and glutamate-rich antigen (GLURP); human papilloma virus (HPV) antigen;human papilloma virus HPV16 antigen; HPV E5 protein; HPV E6 protein; HPVE7 protein; a human immunodeficiency virus (HIV) antigen; and an HIV gagantigen.
 2. The nucleic acid molecule of claim 1 wherein the HSV isselected from the group consisting of HSV-1 and HSV-2.
 3. The nucleicacid molecule of claim 2 wherein the first polypeptide segment comprisesan amino acid sequence selected from the group consisting of: (1) aminoacids 26-265 of SEQ ID NO:27; (2) amino acids 26-265 of SEQ ID NO:29;(3) amino acids 1-244 of the glycoprotein D; (4) amino acids 1-288 ofthe glycoprotein D; and (5) amino acids 1-294 of a mature HSVglycoprotein D with the exception that amino acid 294 is alanine insteadof tryptophan.
 4. The nucleic acid molecule of claim 3 which comprises:nucleotides 76-795 of SEQ ID NO:26; or nucleotides 350-1069 of SEQ IDNO:28.
 5. The nucleic acid molecule of claim 1 which encodes the aminoacid sequence encoded by SEQ ID NO:35.
 6. The nucleic acid molecule ofclaim 1 which comprises a nucleotide sequence selected from the groupconsisting of SEQ ID NO:35; SEQ ID NO:31; SEQ ID NO:32; SEQ ID NO:36;SEQ ID NO:37; and SEQ ID NO:34.
 7. The nucleic acid molecule of claim 1wherein the second polypeptide segment comprises the E5 protein, the E6protein, and the E7 protein.
 8. The nucleic acid molecule of claim 1wherein the fusion protein comprises SEQ ID NO:23.
 9. The nucleic acidmolecule of claim 1 wherein the fusion protein comprises SEQ ID NO:22.10. The nucleic acid molecule of claim 1 which comprises SEQ ID NO:32.11. The nucleic acid molecule of claim 1 wherein the fusion proteincomprises SEQ ID NO:33.
 12. The nucleic acid molecule of claim 1 whichis in a viral vector.
 13. The nucleic acid molecule of claim 1 which isnaked DNA.
 14. The nucleic acid molecule of claim 1 which is in abacterial vector.
 15. The nucleic acid molecule of claim 1 wherein thethird polypeptide segment comprises the transmembrane domain of the HSVglycoprotein D.
 16. A fusion protein encoded by the nucleic acidmolecule of claim
 1. 17. A method of inducing an immune response,comprising providing to a subject in need thereof a fusion protein ofclaim
 16. 18. The method of claim 17 wherein the fusion protein isprovided by administering to the subject a nucleic acid molecule whichencodes the fusion protein.